METHODS OF INDUCING IMMUNITY TO LYME DISEASE AND A COLORIMETRIC ASSAY FOR BORRELIACIDAL ACTIVITY OF ANTISERA

Abstract: A simple, colorimetric borreliacidal assay for the determination of borreliacidal activity of immune serum to Borrelia burgdorferi is disclosed. Also disclosed is a vaccine for Borrelia burgdorferi as well as methods of inducing immunity to bacteria causing Lyme disease, including B. burgdorferi and B. garinii by administering the vaccines of the present invention. Also disclosed are kits comprising one or more vials comprising the elements of the Borrelia burgdorferi vaccine. Also disclosed are vaccine formulations for Lyme disease.

Description: METHODS OF INDUCING IMMUNITY TO LYME DISEASE AND A COLORIMETRIC ASSAY FOR BORRELIACIDAL ACTIVITY OF ANTISERA This is a continuation-in-part (CIP) application of the copending U.S. Patent Application, Serial No. 08/025,379, filed February 26, 1993, the entire disclosure of which is hereby incorporated by reference herein. Applicants claim benefit to the earlier priority date of said U.S. Application, Serial No. 08/025,379, filed February 26, 1993. Field of the Invention The present invention is in the field of medicinal chemistry. In particular, the invention is related to vaccines for Borrelia burgdorferi comprising a saponin, and the use thereof to immunize animals. Background of the Invention Lyme disease is a tick-borne multisystemic disorder characterized by early erythema chronicum migrans, late arthritis, and cardiac and neurologic manifestations in humans and animals (Steere, A.C., N. Engl. J. Med. 527:586-596 (1989); Kornblatt et al, J. Am. Vet. Med. Assoc. 756:960-4 (1985); Lissman et al, J. Am Vet. Assoc. 785:219-20 (1984)). Caused by the spirochete Borrelia burgdorferi (Burgdorfer, W., et al. , Science 267:1317- 1319 (1982)), Lyme disease is the most common tick-borne zoonosis occurring in humans and dogs (Steere, A.C., N. Engl. J. Med. 527:586-96 (1989)). Morbidity of Lyme disease can be as high as 9% in highly endemic area (Alpert et al, NY State J. Med. 92:5-8 (1992)). Thus far, at least three genospecies of Lyme disease spirochetes have been recognized based on genetic and molecular determinants (Baranton et al, Int. J. Sys. Bacteriol. 42:378-83 (1992)). Most North American isolates and partial European isolates are Borrelia burgdorferi sensu stricto. Many European and Asian isolates belong to Borrelia garinii sp. nov. , and group VS461 (Baranton et al, Int. J. Sys. Bacteriol. 42:378-83 (1992); Park et al, J. Clin. Micwbiol. 57: 1831-7 (1993)). Humoral immunity is a major protective mechanism against this bacterial infection or disease (Burgdorfer, W., et al. , Science 267:1317-1319 (1982); Simon, M.M., et al , Immunol Today 72:11-16 (1991); Schaible, U.E., et al , Proc. Natl. Acad. Sci. USA 5:3768-3772 (1990); Fikrig, E. , et al. , Science 250:553-556 (1990)). Convalescent human sera are able to kill the spirochete in the presence of complement in vitro, possibly by altering the bacterial outer membrane to allow the formation of an effective membrane attack complex (Kochi and Johnson, Infect. Immun. 56:314-321 (1988); Kochi, S.K., et al. , J. Immunol. 146:3964-3910 (1991)). Heat-inactivated rat antisera to B. burgdorferi or mouse monoclonal antibody to a surface epitope of the spirochete in the absence of complement are also capable of lysing this spirochete (Pavia, C.S., et al , J. Infect. Dis. 163:656-659 (1991); Coleman and Benach, "Characterization of antigenic determinants of Borrelia burgdorferi shared by other bacteria, " J. Infect. Dis. 765:658-666 (1992)). Furthermore, in vitro borreliacidal activity of hamster immune sera to B. burgdorferi with complement is reported to correlate with in vivo protection (Lovrich, S.D., et al , Infect. Immun. 59:2522-2528 (1991)). Recent studies on Lyme disease have contributed to the better understanding of the pathogenesis of Lyme disease and host immune responses to B. burgdorferi (Garcia-Monco et al, Rheum. Clin. N. Am. 75:711-26 (1989); Szczepanski et al, Micwbiol Rev. 55:21-34 (1991)). Borreliacidal and antiborrelial activity of specific antibody, opsonophagocytosis and subsequent killing constitute the important protective mechanisms against borrelial infection (Benach et al, J. Infect. Dis. 150:491-501 (1984); Kochi et al, Infect. Immun. 56:314-21 (1988); Pavia et al, J. Infect. Dis. 765:656-9 (1991); Ma et al, J. Micwbiol Methods 77:145-53 (1993); Peterson et al, Infect. Immun. 46:608-11 (1984); Schaible et al, Proc. Natl. Acad. Sci. USA 5:3768-72 (1990)). Immunoprotection studies in mice have shown that the outer surface proteins A (OspA) and B (OspB) of B. burgdorferi are protective immunogens and are the candidates for vaccine development (Schaible et al, Proc. Natl. Acad. Sci. USA 5:3768-72 (1990); Fikrig et al, Science 250:553- 6 (1990); Fikrig et al, Infect. Immun. 60:651-661 (1992)). These early observations indicate that protection was possible against B. burgdorferi but not against other species of Borrelia. Canine immune responses to the infection of Lyme disease spirochetes is poorly understood. Low and high antibody responses have been detected in the spirochete-positive and asymptomatic dogs (Kornblatt et al, J. Am. Vet. Med. Assoc. 186:960-4 (1985); Lissman et al, J. Am. Vet. Assoc. 185:219-20 (1984); Burgess, E.C., Lab. Ani. Sci. 56:288-90 (1986)). Antibody response to OspA were rarely detectable in naturally exposed dogs. (Greene et al, J. Clin. Micwbiol 26:648-53 (1988)). This observation is similar to the serological findings in human Lyme disease patients that antibodies to OspA and OspB are rarely detectable even at late stage of Lyme disease (Craft et al, J. Clin. Invest. 78:934-39 (1986); Coleman et al, J. Infect. Dis. 755:756-65 (1987)). Thus, whether or not OspA and OspB based vaccine would stimulate protective immune responses in Lyme disease hosts needed to be addressed. Borreliacidal activity of immune sera is usually determined by an in vitro assay adapted from an antileptospiral assay (Kochi and Johnson, Infect. Immun. 56:314-321 (1988)). Because B. burgdorferi does not reliably form isolated colonies on currently available agar medium, spirochete killing by the bactericidal antibody is determined by dark-field microscopy on the basis of loss of motility , refractility , or extensive surface blebbing (Kochi and Johnson, Infect. Immun. 56:314-321 (1988); Kochi, S.K., et al. , J. Immunol. 146:3964- 3970 (1991); Lovrich, S.D., et al , Infect. Immun. 59:2522-2528 (1991); Callister, S.M., et al , J. Clin. Micwbiol 29:1773-1776 (1991)). Enumeration of live spirochetes, however, is difficult since antibody specific to the outer surface proteins or lysate of B. burgdorferi strongly agglutinates the spirochetes (Coleman and Benach, "Characterization of antigenic determinants of Borrelia burgdorferi shared by other bacteria," J. Infect. Dis. 765:658-666 (1992); Ma, J., et al. , "Antibody mediated microagglutination of Borrelia burgdorferi correlates with antibody titers to rec OspA," V Int. Con Lyme Borreliosis, Arlington, VA, USA (1992)). This agglutination also makes it difficult to examine the viability of the agglutinated bacteria. To confirm the observation obtained by dark-field microscopy, a radioactive incorporation assay or spirochete culture is conducted as well (Kochi and Johnson, Infect. Immun. 56:314-321 (1988); Pavia, C.S., et al. - J. Infect. Dis. 163:656-659 (1991); Lovrich, S.D., et al , Infect. Immun. 59:2522-2528 (1991); Callister, S.M., et al , J. Clin. Micwbiol 29:1773-1776 (1991)). The current borreliacidal assays, therefore, are tedious and time-consuming, and lack reliability. There is also a safety issue surrounding the use of radioactive materials and observing the pathogenic spirochete under a dark- field microscope. Summary of the Invention The present invention is directed to a new colorimetric borreliacidal assay (CBA) for determination of the bactericidal activity of antiserum to B. burgdorferi, which is simple, reliable, and exhibits an easily measurable end-point. The results of the CBA correlated well with those of both direct dark-field microscopy and [ 3 H]-thymidine incorporation assay (TIA). This colorimetric microtiter assay was measured by a microplate reader and thus the results can be processed automatically using an appropriate computer program. In particular, the invention relates to a method for the determination of the bactericidal activity of an antiserum to B. burgdorferi, which comprises (a) contacting said antiserum,with a sample containing B. burgdorferi and a suitable color pH indicator such as phenol red, for measurement of bacterial growth; and (b) measuring the absorbance of the sample; wherein high absorbance, when compared to a control sample which does not contain B. burgdorferi, is an indication of high bactericidal activity. Color pH indicators are known in the art and may be readily obtained from the various suppliers. For example, various color pH indicators are described in the 1993 catalogue of Sigma Chemical Co., St. Louis, Missouri, at page 1438. The invention also relates to a vaccine, comprising OspA, OspB or fragments thereof; and a saponin adjuvant. More specifically, the invention relates to a vaccine, comprising full- length lipoproteins OspA and/or OspB; and a saponin adjuvant such as QS-21. The invention also relates to a method of inducing immunity to bacteria causing lyme disease, e.g., B. burgdorferi and B. garinii, in an animal, comprising administering to the animal a vaccine comprising OspA, OspB or fragments thereof; and a saponin adjuvant. More specifically, the invention relates to a method of inducing immunity to B. burgdorferi and other species of the genus Borrelia in an animal, comprising administering to the animal a vaccine comprising full- length lipoproteins OspA and/or OspB; and a saponin adjuvant such as QS-21. An advantage of the present invention is that the immunogenicity of OspA- and OspB-based subunit vaccine is greatly enhanced by using the full- length lipoproteins OspA and OspB. A further advantage of the present invention is that the immunogenicity of Osp subunit vaccine is further enhanced by the adjuvant QS-21. Another advantage of the present invention is that a preferred vaccine formulation comprising OspA, OspB, and QS-21 displays borrelicidal activity against not only the homologous and closely related strains, but also against the heterologous and different genospecies of lyme disease spirochetes as well as other species of the genus Borrelia. Moreover, the OspA- and OspB-based vaccine of the present invention elicits synergistically higher functional humoral immune response than a single protein-based vaccine. Description of the Figures Figure 1 depicts the colorimetric borreliacidal assay of mouse antisera to B. burgdorferi strain B31 against the homologous strain. Borrelial culture at logarithmic growth phase were centrifuged for 8 min. at 9000 x g at 15°C and resuspended in fresh mBSK. Ninety five μl of the borrial suspension (containing approximately 4 x 10 6 of spirochetes) and 5 μl of guinea pig complement were mixed with an equal volume of the serially diluted (column 1 to 12) heat-inactivated mouse antisera (row A to C), normal sera (row D to F), and mBSK control row (row G to H) in mBSK containing 120 μg of phenol red in a microtiter plate, and incubated for 48 h at 32°C. Red color (dark) indicates borrelial death; yellow color (light) represents borrelial survival and growth. Figure 2 depicts a graph showing the correlation of the colorimetric assay with the pHlthymidine incorporation assay. One hundred μl of B. burgdorferi strain B31 (containing about 8 x 10 6 spirochetes) was serially diluted in mBSK in 96- ell plates, and incubated with an equal volume of mBSK-PR for 30 h at 32°C. Twenty μl of pH]thymidine in mBSK (2 μCi) were added to each well and the plates were incubated for another 18 h for pulse-labelling the live spirochete (Pavia, C.S. et al, J. Infect. Dis 163:656- 659 (1991)). The absorbance at 562/630 nm (open circles) was measured by a microplate reader, and the radioactivity (closed circles) measured as counts per minutes by a scintillation counter. As shown in the figure, the results of CBA correlated with those of TIA (R = 0.977). The error bars represent the standard deviation of 3 measurements. Figure 3 depicts a graph showing the changes of absorbance at 562/630 nm of the colorimetric borreliacidal assay with mouse antisera to OspA-B31 against the homologous strain. The assay was done as described in the description of Figure 1. The plate was incubated at 32°C for 120 h, and the absorbance measured using a microtiter reader before and every 24 h after incubation. Each serum dilution was performed in triplicate. The error bars represent the standard deviation of 3 measurements. Figure 4 depicts a graph showing the significant inhibition of the decrease in absorbance at 562/630 nm of the colorimetric borreliacidal assay by mouse antisera to B. burgdorferi stain B31. The serially diluted, heat- inactivated antisera or normal sera were incubated with the strain B31 in the presence of complement as described above in the description of Figure 1. Each serum dilution was performed in triplicate. The absorbance was measured after 48 h incubation. High absorbance indicates borrelial death; the low absorbance represents borrelial survival and growth. The error bars represent the standard deviation of 3 measurements. Figure 5 depicts a graph showing the average borreliacidal activity of canine antisera to truncated OspA and/or OspB derived from B. burgdorferi strain B31 against the homologous strain. Figure 6 depicts a graph showing the average borreliacidal activity of canine antisera to truncated OspA and/or OspB derived from B. burgdorferi strain B31 against the heterologous California strain CA-2-87. Figure 7 depicts a graph showing the borreliacidal activity of canine antisera to truncated OspA and/or OspB derived from B. burgdorferi strain B31 against the homologous strain. Figure 8 depicts a graph showing the borreliacidal activity of canine antisera to truncated OspA and/or OspB derived from B. burgdorferi strain B31 against the heterologous California strain CA-2-87. Figure 9 depicts a graph showing the borreliacidal activity of C3H/Hej female mice that had been immunized twice with either 25 μg of truncated OspA, 25 μg of truncated OspB, or 25 μg of truncated OspA and 25 μg of truncated OspB. Figure 10 depicts a graph showing the borreliacidal activity of C3H/Hej female mice that had been immunized twice with either 25 μg of truncated OspA + 20 μg of QS21, 25 μg of truncated OspB + 20 μg of QS21, or 25 μ of truncated OspA and 25 μg of truncated OspB + 20 μg of QS21. Figure 11 depicts a fluorograph of nitrocellulose membrane showing radiolabeling of FLOspA and FLOspB by [9,10- 3 H]palmitic acid. Escherichia coli strain MZ-1 harboring ospA or ospB gene was grown in LB broth to log phase at 32 °C. [9, 10- 3 H]palmitic acid was added to the culture and incubated for 2 h at 42 °C. Bacteria were lysed, and OspA and OspB precipitated using specific MAbs described in the specification, by modification of the procedure (Katona et al, Infect. Immun. 60:4995-5003 (1992)). Samples were boiled for 5 min, subjected to SDS-PAGE, and transferred to nitrocellular membrane. The membrane was then treated with En 3 Hance spray (Dupont- NEN, Boston, MA) and exposed to film. 1, FLOspA; 2, TOspA; 3, FLOspB; 4, TOspB. Molecular weight markers in KDa. Figure 12 depicts antibody isotype titers of canine antisera to various experimental vaccines. Beagles at age of 12 and 16 weeks were immunized subcutaneously twice with various experimental vaccines. Immune sera were isolated two weeks after last immunization, and assayed by ELISA using plates coated with B. burgdorferi antigens (Cambridge Biotech Corporation, Worcester, MA; Lindenmayer et al, J. Clin. Micwbiol 28:92-6 (1990)). Antibody isotypes were determined using isotype-specific goat anti-dog IgG conjugated to horseradish peroxidase (Bethyl Laboratories, Inc., Montgomery, TX). The titer was defined as the highest serum dilutions resulting in an absorbance value 1.0. The error bars stand for standard error. Number of immune serum sample from top to bottom: 8, 10, 16, 5, 5, 5. Figure 13 depicts representative patterns of immunoblot with naturally exposed dog sera and antisera to experimental vaccines. Naturally exposed dog sera were isolated in New York area. Antisera to experimental vaccine containing 25 μg each FLOspA and FLOspB and 50 μg QS-21 were prepared as described above with respect to Fig. 12. Immunoblot was performed with antigen strips according to the instruction of manufacturer (Cambridge Biotech Corporation, Worcester, MA). Serum samples at dilution of 1:50 and goat anti-dog IgG F(ab') 2 conjugated onto horseradish peroxidase (Bethyl Laboratories, Inc., Montgomery, TX) were used to detect the specific antibody. Lane 1, positive serum; lanes 2 to 12, naturally exposed dog sera; lane 13 to 23, antisera; lane 24, negative serum. Figures 14A and 14B depict comparison of OspA and OspB specific antibody isotype titer of naturally exposed canine sera and antisera to experimental vaccines. Naturally exposed dog sera were described above with respect to Fig. 13. Antisera to experimental vaccine containing 25 μg each of FLOspA and FLOspB and 50 μg of QS-21 were described above with respect to Fig. 12. ELISA was done using plates coated with 0.2 μg OspA and 0.2 μg OspB, respectively. Antibody isotypes were determined using isotype-specific goat anti-dog IgG conjugated to horseradish peroxidase (Bethyl Laboratories, Inc., Montgomery, TX). Titer was defined as described above with respect to Fig. 12. Figure 15 depicts antiborrelial activity against B. burgdorferi sensu stricto strains B31 and CA-2-87 of canine antisera to vaccines formulated with lipidated or nonlipidated OspA and OspB and QS-21. Antisera are described above with respect to Figure 12. Antiborrelial activity of antisera was determined as described in Table 1. Error bars represent the standard deviation of measurements of 8, 10, and 16 serum samples, respectively (from top to bottom). Figure 16 depicts antiborrelial activity against different geographic Lyme disease spirochetes of B. burgdorferi sensu stricto and B. garinii sp. nov. of canine antisera to experimental vaccine formulated with 25 μg each of FLOspA and/or FLOspB and with or without QS-21. Antisera preparations are described above with respect to Fig. 12. Antiborrelial activity of antisera was tested as described in Table 1. Error bars represent the standard deviation of measurements of 32, 10, 10, 10, and 3 serum samples, respectively (from top to bottom of legend). High absorbance indicates high antiborrelial activity. Fϊgure 17 depicts correlation of antiborrelial activity of canine antisera with isotypes IgGl and IgG2 antibody titer. Antisera to experimental vaccine containing 25 μg each of FLOspA and FLOspB and 50 μg QS-21 are described above with respect to Fig. 12. Antiborrelial activity and IgG antibody titers were determined as described in Table 1 and Fig. 12, respectively. Antiborrelial activity of antisera correlated with IgG2 antibody titer (R=0.61) and not with IgGl (R=0.13). Figure 18 depicts expression of recombinant OspA and OspB in E. coli strain MZ-1. Bacteria containing recombinant plasmids encoding OspA or OspB were grown to logarithmic phase at 32 °C, and then incubated at 42 °C for 1 h for induction of recombinant protein synthesis. Whole cell lysates of bacteria before and after induction were analyzed by SDS-PAGE, and visualized by Coomassie blue staining. 1 , before induction of MZ-1 harboring pLCBCl (Beltz, G. A. et al , U.S. Patent No. 4,753,873 (1988)) and ospA; 2, after induction; 3, purified OspA; 4, after induction of MZ-1 harboring pLCBCl and ospB; 5, purified OspB. Molecular weight markers (Bio-Rad Laboratories, Melville, New York) in KDa. Figures 19 A, 19B and 19C depict SDS-PAGE and immunoblotting of whole cell lysates of B. burgdorferi. Four to 6 μg of lysates of the bacteria were analyzed for protein composition on the 11 % SDS-PAGE gel, and visualized by Coomassie blue staining (Fig. 19A). The antigenic properties of OspA and OspB were tested by transferring the separated proteins to the membrane and probing with 1:20 dilution of mouse antisera to OspA (Fig. 19B) or OspB (Fig. 19C) of strain B31. 1, strain B31; 2, strain CA-2- 87; 3, strain Fr; 4, strain G25. → indicates an approximately 22 KDa protein band in Fig. 19C. Molecular weight markers are in KDa. Figure 20 depicts antibody isotype titers of mouse antisera to OspA and OspB formulated with or without either QS-21 or alum. Serially diluted mouse antisera were assayed by ELISA as described (Lindenmayer J. et al. , J. Clin. Microbiol 5:92-96 (1990)). The isotype-specific goat anti-mouse IgG conjugated to horseradish peroxidase was used to measure antibody isotype. The bars represent group means. Figure 21 depicts OspA and OspB protein coding regions within B. burgdorferi. Figure 22 depicts the basic features of the expression vector used to express B. burgdorferi recombinant OspA and OspB antigens. Figure 23 depicts the entire DNA sequence of pLCBC10spA8+6 and the origin of each base. The sequence information was obtained from the following publicly available sources: 1. pBR322 from Genebank Accession # J01749 2. lambda from Genebank Accession # J02459 3. OspA/B from Bergstrom et al. , Mol. Microbiol. 5:479- 486 (1989). Figure 24 depicts the entire DNA sequence of pLCBC10spB8+4 and shows the origin of each base. The sequence information was obtained from the following publicly available sources: 1. pBR322 from Genebank Accession # J01749 2. lambda from Genebank Accession # J02459 3. OspA/B from Bergstrom et al. , Mol Microbiol 5:479- 486 (1989). Figure 25 depicts a restriction fragment profile of the OspA clone pLCBC10spA8+6. From left to right, each lane contains either a control marker or pLCBC10spA8+6 incubated with the indicated restriction enzyme(s). Lane 1: markers λHindlll + XHaelll; lane 2: uncut; lane 3: BamHI; lane 4: PvuII; lane 5: EcoRI; lane 6: Hindlll; lane 7: Ndel; lane 8: Pstl; lane 9: Seal; lane 10: EcoRI + PvuII; lane 11: EcoRI + Hindlll; lane 12: Pstl + PvuII; lane 13: λHindlll + XHaeffi markers. Figure 26 depicts a restriction fragment profile of the OspB clone pLCBC10spB8+4. From left to right each lane contains either a control marker or pLCBC10spB8+4 incubated with the indicated restriction enzyme(s). Lane 1: markers λHindlll + φXHaelll; lane 2: uncut; lane 3: BamHI; lane 4: EcoRI; lane 5: Ndel; lane 6: Pstl; lane 7: PvuII; lane 8: BamHI + EcoRI; lane 9: EcoRI + Hindlll; lane 10: EcoRI + Pstl; lane 11: Hindlll + Sspl; lane 12: PvuII + Sspl; lane 13: markers λHindlll + XHaelll. Description of the Preferred Embodiments The present invention relates to a method for the determination of the bactericidal activity of an antiserum to B. burgdorferi, which comprises (a) contacting said antiserum with a sample containing B. burgdorferi and a suitable color pH indicator, such as phenol red, for measurement of bacterial growth; and (b) measuring the absorbance of the sample; wherein high absorbance, when compared to a control sample which does not contain said antiserum is an indication of high bactericidal activity. Color pH indicators are known in the art and may be readily obtained from the various suppliers. For example, various color pH indicators are described in the 1993 catalogue of Sigma Chemical Co., St. Louis, Missouri, at page 1438. The borreliacidal activity of immune sera to B. burgdorferi is an important parameter in the in vitro evaluation of protective efficacy of vaccines against Lyme disease (Simon, MM., et al. , Immunol. Today 12: 11- 16 (1991); Schaible, U.E., et al , Proc. Natl. Acad. Sci. USA 5:3768-3772 (1990); Kochi and Johnson, Infect. Immun. 56:314-321 (1988); Lovrich, S.D. , et al , Infect. Immun. 59:2522-2528 (1991); Callister, S.M., et al , J. Clin. Microbiol. 29:1773-1776 (1991)). Currently used borreliacidal assays examine only a small portion of the sample for viable spirochetes under a dark-field microscope (Kochi and Johnson, Infect. Immun. 56:314-321 (1988); Kochi, S.K., et al. , J. Immunol. 146:3964-3910 (1991); Lovrich, S.D., et al , Infect. Immun. 59:2522-2528 (1991); Callister, S.M. , et al , J. Clin. Microbiol. 29:1773-1776 (1991)). Radioactive incorporation assays and spirochete culture are used to verify the results (Kochi and Johnson, Infect. Immun. 56:314-321 (1988); Pavia, C.S. , et al , J. Infect. Dis. 163:656-659 (1991); Lovrich, S.D., et al , Infect. Immun. 59:2522-2528 (1991); Callister, S.M. , et al. , J. Clin. Microbiol 29: 1773-1776 (1991)). It is apparent that the existing bactericidal assays lack simplicity, reliability, and cannot handle a large number of serum samples. The present microtiter CBA uses phenol red as an indicator of the accumulation of nonvolatile acid generated by spirochete metabolism. The CBA detects borreliacidal activity of immune sera by measuring the absorbance at 562/630 nm. Significant decrease in absorbance represents the bacterial survival and growth; a small decrease in absorbance indicates borrelial killing. To increase the sensitivity of the colorimetric assay, a relatively high concentration of phenol red (final concentration 60 μg/ml) was required. The dual wavelengths of 562/630 nm most sensitively reflected the absorbance changes in the presence of acid. To evaluate the validity of the colorimetric assay, both the TIA and colorimetric assay using serially diluted spirochetes or using a concentration of 4 x 10 6 spirochetes and serially diluted antisera to lysates of strain B31 were performed. Correlation coefficients of these two assays were 0.977 and 0.935, respectively, and were linearly related over the useful range of most microplate readers. An increase in the absorbance of the first several serum dilutions after 24 h incubation, compared with the absorbance before incubation, was consistently seen (Fig. 3). This change could have been caused by the dissociation of CO 2 from the plate during the incubation. The slow growth of the spirochete and borreliacidal activity of the antisera might have prevented the production of the enough acid products to reduce pH. The absorbance of the borreliacidal mixture with each serum dilution decreased at a constant rate during 48 h to 96 h incubation (Fig. 3). After 48 h incubation, the inhibition of borrelial growth by the antisera was clearly detectable, as shown by the absorbance. The inhibition of the borrelial growth (significantly less absorbance change) was clearly dependent on the concentration of antisera (Figs. 3 and 4). The mouse antisera to lysates of strain B31 had a high borreliacidal activity against the homologous strain B31 (Fig. 4), strains Cr (a Wisconsin tick isolate), and Fr (a German tick isolate), but had little borreliacidal activity against the Swedish strain G25, a different genomic species in terms of rRNA gene restriction patterns (Postic, D. et al, Res. Micwbiol 141:465-415 (1990)). Similarly, the antisera to strain G25 possessed a high borreliacidal activity against the homologous strain G25, but did not against other strains tested. Normal mouse sera, compared with mBSK control, were not borreliacidal (Fig. 4). Dark-field microscopy showed that the spirochetes were killed or lysed by the antisera, but not by the normal mouse sera. Thus, the inhibition of absorbance changes in the CBA was because of the borreliacidal activity of the specific antibody. The turbidity increase due to the borrelial growth was negligible and did not significantly influence the absorbance changes. In the presence of a high concentration of the antisera, approximately 10-20% of the spirochetes still survived. This observation was consistent with previous reports that human convalescent sera or rat immune sera fail to kill all spirochetes in vitro (Pavia, C.S., et al , J. Infect. Dis. 163:656-659 (1991); Lovrich, S.D., et al , Infect. Immun. 59:2522-2528 (1991); Callister, S.M., et al , J. Clin. Microbiol 29:1773-1776 (1991)). Both the CBA and TIA showed the antisera to strain B31 without complement killed the spirochete as efficiently as those containing guinea pig complement. Human or hamster immune sera, however, do require complement for killing B. burgdorferi (Kochi and Johnson, Infect. Immun. 56:314-321 (1988); Kochi, S.K., et al , J. Immunol. 146:3964-3910 (1991); Lovrich, S.D., et al , Infect. Immun. 59:2522-2528 (1991)), while heat- inactivated rat antisera to B. burgdorferi or monoclonal antibody to a surface epitope of the spirochete in the absence of complement source are also borreliacidal (Pavia, C.S., et al. , J. Infect. Dis. 163:656-659 (1991); Coleman and Benach, "Characterization of antigenic determinants of Borrelia burgdorferi shared by other bacteria," J. Infect. Dis. 765:658-666 (1992)). Classically, complement components are required for the destruction of the Gram-negative bacteria, including B. burgdorferi, by bactericidal antibody. To quantitate the borreliacidal activity of immune sera, the borreliacidal titer was defined as the highest dilution of sera which inhibits absorbance change caused by 50% (2 x 10 6 bacteria) of spirochetes in the CBA. In summary, the microtiter CBA for determination of borreliacidal activity of immune serum has been developed with the advantages of simplicity, reliability, and safety. Furthermore, the CBA can handle a large number of serum samples and is valuable in Lyme vaccine development and as a diagnostic method for Lyme borreliosis. Thus, the invention also relates to a method for the detection of Lyme borreliosis, comprising (a) contacting an antiserum against B. burgdorferi with a suitable color pH indicator, such as phenol red, for measurment of bacterial growth, and a sample suspected of containing B. burgdorferi; and (b) measuring the absorbance of the sample; wherein high absorbance, when compared to a control sample which does not contain B. burgdorferi, is an indication that B. burgdorferi is present in said suspected sample. Color pH indicators are known in the art and may be readily obtained from the various suppliers. For example, various color pH indicators are described in the 1993 catalogue of Sigma Chemical Co., St. Louis, Missouri, at page 1438. The invention also relates to a vaccine, comprising OspA, OspB or fragments thereof; and a saponin adjuvant. Investigation of immunoprotective antigens of Lyme disease spirochetes and development of Lyme disease vaccine have been the focus of Lyme disease research (Fikrig et al, Science 250:553-6 (1990); Fikrig et al, Infect. lmmun. 60:651-661 (1992); Edelman, R. , Vaccine 9:531-2 (1991); Fikrig et al, Proc. Natl Acad. Sci. USA 59:5418-21 (1992)). OspA or OspB based experimental vaccines formulated with Freund's adjuvants have been shown to confer protection against challenge by either syringe injection or spirochete infected tick feeding in mouse model (Fikrig et al. , Infect. Immun. 60:651-661 (1992); Fikrig et al, Proc. Natl. Acad. Sci. USA 59:5418-21 (1992)). Although these results are encouraging, it is important to determine the influence of clinically acceptable adjuvants on the immunogenicity of the recombinant proteins. Hence, the inventors have identified and purified a potent saponin adjuvant, QS-21, from Quillaja saponaria (Kensil, C. R. et al. , J. Immunol. 146:431-431 (1990)). This adjuvant significantly enhances both humoral and cellular immune responses to a variety of antigens and has been used in recombinant subunit vaccines including the experimental HIV-1 gpl60 protein vaccine and commercially available feline leukemia virus vaccine (Marciani D. J. et al , Vaccine 9:89-96 (1991); Newman, M. J. et al , J. Immunol 148:2351-2362 (1992); Wu, J.-Y. et al , J. Immunol. 745:1519-1525 (1992)). The impact of QS-21 and aluminum hydroxide (alum) on the functional antibody responses to the recombinant OspA and OspB derived from B. burgdorferi strain B31 was determined in mice. Only QS-21 induced high titers of IgG2a and IgG2b antibodies, the complement fixing isotypes (Kochi, S. K. et al , J. Immunol 146:3964-3910 (1991); Schmitz, J. L. et al , Infect. Immun. 60:2677-2682 (1992); Spiegelberg, H. L., Adv. Immunol 79:259-294 (1974)). QS-21 was shown to be superior to alum in enhancing functional antibody response to OspA and OspB. The experimental vaccine containing OspA and OspB formulated with QS-21 conferred complete protection against infection with either the homologous or the heterologous strains of B. burgdorferi in mice. Hence, according to the obtained results a saponin adjuvant, QS-21, significantly enhances immunogenicity of OspA and OspB. In contrast, aluminum hydroxide, a widely used adjuvant for human vaccines, does not significantly influence OspA or OspB based experimental Lyme vaccines. As mentioned above, the impact of the adjuvants QS-21 and aluminum hydroxide (alum) on the immunogenicity of recombinant outer surface protein A (OspA) and B (OspB) of Borrelia burgdorferi was investigated. Both nonacylated OspA and OspB derived from strain B31 were expressed in Escherichia coli and purified by reversible citraconylation and anion exchange chromatography . Antisera to OspA or OspB were prepared in mice with antigens formulated with QS-21 or alum, and evaluated for specific immunoglobulin G isotypes, agglutination, and borreliacidal activity. QS-21 significantly enhanced IgG2a and IgG2b antibody responses to OspA and OspB, and IgGl response to OspA when compared with the formulation containing antigen alone. In contrast, alum significantly inhibited the induction of IgG2a and IgG2b responses to OspA. Alum had no significant effect on IgGl response to OspA, or IgG2a and IgG2b responses to OspB, but significantly enhanced IgGl antibody response to OspB. Antisera to OspA or OspB formulated with QS-21 possessed higher titers of agglutinating antibody than antisera to OspA or OspB alone, respectively. Borreliacidal activity was 8- to 64-fold higher in antisera to OspA formulated with QS-21 than in antisera to OspA formulated with or without alum. These antisera were highly borreliacidal to B. burgdorferi, New York strain B31, a California isolate CA-2-87, German isolate Fr, and Swedish J garinii species, G25. Antisera to OspB formulated with QS-21 were highly borreliacidal to strains B31 and Fr, but not to CA-2-87 and G25, Antisera to OspB formulated with alum were borreliacidal only to B31. Thus, OspA was superior to OspB and QS-21 superior to alum at eliciting functional antibody responses. The vaccine containing OspA and OspB formulated with QS-21 was protective in mice against infection with 10 s infectious spirochetes of strains B31 or CA-2-87. As mentioned above, experimental vaccines containing OspA and Freund's adjuvant can also eliminate B. burgdorferi from challenge ticks feeding on the immunized mice (Fikrig, E. et al. , Proc. Natl. Acad. Sci. USA 59:5418-5421 (1992)). It has been shown that in vivo immunoprotection is correlated with in vitro borreliacidal activity of immune serum to B. burgdorferi (Lovrich, S. D. et al , Infect. Immun. 59:2522-2528 (1991)). As part of the present invention, we studied and, hence, determined the role of the adjuvants QS-21 and alum on enhancement of functional antibody responses to recombinant OspA and OspB of B. burgdorferi. Both recombinant OspA and OspB were not acylated and were freely soluble proteins. They were monomeric at the concentrations used. The dose of QS-21 used in the vaccine formulation was below its critical micellar concentration (Kensil, C. R. et al , Vaccine Res. 2:273-281 (1993). The possibility exists that these antigens and QS-21 formed soluble lipid-protein complexes. This is fundamentally different from the antigens adsorbed onto an insoluble matrix such as alum. Mice immunized with Osp vaccine formulated without adjuvant raised significantly higher titers of antibody to OspA than to OspB, indicating OspA was more immunogenic than OspB. In general, mice immunized with OspA or OspB formulated with QS-21 raised significantly higher antibody responses than those immunized with the antigens alone or antigens formulated with alum. These antisera also possessed higher agglutination antibody titers and borreliacidal activity. It appeared that the borreliacidal activity of the antisera was correlated with the agglutination antibody titer and ELISA antibody titer with the exception of the low agglutination titer with strain G25. Antisera to OspA formulated with QS-21 had a higher titer of agglutinating antibody than antisera to OspB formulated with QS-21. Those antisera reacted with not only strain B31, but also the heterologous California isolate CA-2-87, German isolate Fr, and Swedish isolate G25. Similarly, antisera to OspA formulated with QS-21 had significantly higher borreliacidal activity against the 4 strains than antisera to OspB formulated with QS-21. These results showed that OspA induced higher functional antibody responses than OspB. In addition, antisera to OspB formulated with QS-21 also recognized an approximately 22 KDa protein band of strains B31, CA-2-87, and Fr, but not strain G25. This OspB related lower molecular mass protein may be similar to a 21 KDa protein that Bundoc and Barbour have previously observed (Bundoc & Barbour, Infect. Immun 57:2733-2741 (1989)). Recently, a premature stop codon in the ospB gene that terminates OspB 58 amino acids short of the full length protein has been observed by Rosa et al. (Rosa, P. A. et al , Mol. Microbiol 6:3031-3040 (1992)). They indicate that this lower molecular mass protein may be a smaller OspB fragment. SDS-PAGE and immunoblotting analyses showed that OspA and OspB of strain G25 and OspB of the California isolate CA-2-87 were different from those of strain B31 and the German isolate Fr based on their migration rate. However, antisera to B31 OspA and OspB reacted with OspA and OspB of strains G25, CA-2-87, and Fr. This observation was similar to a previous report that antiserum to recombinant OspA of strain B31 reacts with OspA proteins of other strains including some European strains (Milch & Barbour, J. Infect. Dis. 760:351-353 (1989)). Recently, the strains B31 and G25 were designated as OspA serotypes 1 and 6, respectively, based on an OspA serotyping system (Wilske, B. et al. , J. Clin. Microbiol 57:340-350 (1993)). Three genospecies of borrelial isolates have been proposed; strains B31 and G25 are included in B. burgdorferi sensu stricto and B. garinii sp. nov. , respectively (Milch, L. et al , J. Infect. Dis. 760:351-353 (1989)). QS-21 significantly increased the agglutination antibody and borreliacidal activity against borrelial isolates with different OspA serotypes or genospecies. This functional increase in antisera may have resulted from a broadening of antibody response to outer surface exposed epitopes on OspA and OspB. This should be kept in mind when developing an effective Lyme disease vaccine because at least seven different OspA serotypes of B. burgdorferi have recognized thus far (Wilske, B. et al , J. Clin. Microbiol 57 :340-350 (1993)). The major antibody isotypes enhanced by QS-21 in mice were IgG2a and IgG2b. Alum increased only the IgGl antibody response, consistent with a previous report (Byars, N. E. et al , Vaccine 9:309-318 (1991)). This characteristic of QS-21 in enhancing IgG isotypes is important because the isotypes of IgG antibody differ in immunoprotective efficacy for many infectious diseases (Spiegelberg, H. L., Adv. Immunol. 79:259-294 (1974); Briles, D. E. et al , J. Mol. Cell. Immunol. 7:305-309 (1984); Coutelier, J. P. et al. , J. Exp. Med. 765:2373-2378 (1988)). Complement fixation (Kochi, S. K. et al. , J. Immunol. 146:3964-3910 (1991); Schmitz, J. L. et al , Infect. Immun. 60:2677-2682 (1992); Spiegelberg, H. L., Adv. Immunol. 79:259-294 (1974)); and efficient opsonization by IgG2a and IgG2b (Benach, J. L. et al. , J. Infect. Dis. 150:491-501 (1984); Unkeless & Eisen, J. Exp. Med. 742: 1520-1533 (1975)) may enhance protective mechanisms against borrelial infection. Human or hamster immune sera require complement for killing B. burgdorferi (Kochi, S. K. et al. , J. Immunol. 146:3964-3910 (1991); Schmitz, J. L. et al. , Infect. Immun. 60:2677-2682 (1992)), while mouse OspB-specific monoclonal antibody IgGl can also kill spirochetes in the absence of complement (Coleman, J. L. et al , Infect. Immun. 60:3098-3104 (1992)). Thus, B. burgdorferi may be killed in both complement-dependent and complement-independent fashions. It has been reported, however, that OspA- specific monoclonal antibody IgG2b confers better passive protection against experimental diseases than OspA-specific monoclonal antibody IgGl (Schaible, U. E. et al , Pwc. Natl. Acad. Sci. USA 5:3768-3772 (1990)). Hamster IgG2 from immune sera conferred complete passive protection to irradiated hamsters from experimental borrelial infection in a complement-dependent fashion (Schmitz, J. L. et al , Infect. Immun. 60:2677-2682 (1992)). Thus, complement-mediated killing may be a significant and efficient protection mechanism in vivo. QS-21 also significantly induced OspA and OspB specific antibody IgGl and IgG2 responses in dogs and the borreliacidal activity appeared to be associated with IgG2. Thus, QS-21 serves as an important component in a vaccine against Lyme disease. OspA or OspB formulated with Freund's adjuvant (Fikrig, E. et al. , Science 250:553-556 (1990); Fikrig, E. et al , Infect. Immun. 60:651-661 (1992); Fikrig, E. et al , Proc. Natl. Acad. Sci. USA 59:5418-5421 (1992)) or OspA formulated with alum (Erdile, L. F. et al , Infect. Immun. 67:81-90 (1993)) have been shown to confer protection against experimental challenge in mice. The present study indicated that QS-21 was more potent than alum in enhancing the immunogenicity of OspA and OspB. Antibody to OspA formulated with QS-21 was borreliacidal to all the strains tested, and antibody to OspB formulated with QS-21 borreliacidal to strains B31 and Fr. Some borrelial isolates do not produce OspA or OspB. Thus, the vaccine containing both OspA and OspB may provide better and broader protection. Our in vivo study showed that the vaccine containing OspA, OspB, and QS-21 was capable of conferring protection against the infection of the high doses of die homologous and heterologous spirochetes. QS-21 has been used in a commercially available feline leukemia virus vaccine (Marciani, D. J. et al , Vaccine 9:89-96 (1991)) and in an experimental HIV-1 vaccine in rhesus macaques (Livingston, P. O., Annals New York Acad. Sci. 690:204-213 (1993)). This adjuvant has been shown to significantly enhance both antibody and cell-mediated immune responses and has little or no toxicity in laboratory animals (Kensil, C. R. et al , J. Immunol 146:431-431 (1990); Marciani D. J. et al. , Vaccine 9:89-96 (1991); Newman, M. J. et al. , J. Immunol 745:2357-2362 (1992); Livingston, P. O., Annals New York Acad. Sci. 690:204-213 (1993)). A human phase I melanoma ganglioside vaccine trial has shown that a QS-21 formulated GM2- KLH vaccine induces significantly higher antibody responses than other adjuvant formulated vaccines without associated toxicity. Therefore, QS-21 is a useful adjuvant for both veterinary and human Lyme vaccine. In summary, OspA and OspB of B. burgdorferi possessed borreliacidal epitopes, and the adjuvant QS-21 was more efficient than alum in inducing antibody responses to OspA and OspB. QS-21 significantly enhanced IgG2a, IgG2b, and functional antibody responses, and as shown below, it is an important component of a Lyme disease vaccine. The experimental vaccine containing OspA and OspB formulated with QS-21 was shown to be highly protective in mice and as shown below, was found to be highly effective in the prevention of Lyme disease in dogs. TABLE 1. Agglutination with divergent borrelial strains of mouse antisera to OspA and OspB formulated with or without QS-21 Agglutination titer 1 Immunogens Strain B31 CA-2-87 G25 Fr OspA 1 ,600 800 < 50 400 OspA + alum 800 800 100 800 OspA + QS-21 6,400 1,600 400 1,600 OspB 200 100 ≤ 50 100 OspB + alum 400 400 ≤ 50 400 OspB + QS-21 400 200 < 50 400 Normal mouse serum < 50 ≤ 50 < 50 < 50 1 Microagglutination of mouse antisera to OspA or OspB with different geographic isolates of B. burgdorferi was performed by incubating 100 μl of B. burgdorferi (approximately 1 x 10 6 spirochetes) with an equal volume of heat-inactivated, serially diluted antisera in mBSK medium at 32 °C for 2 hours. Agglutination was determined by dark-field microscopy, and its titer defined as the highest dilution of the sera that caused ≥ 50% of the spirochetes to agglutinate. TABLE 2. Borreliacidal Activity against divergent borrelial strains of mouse antisera to OspA and OspB formulated with or without adjuvants Borreliacidal titer 1 Immunogens Strain B31 CA-2-87 G25 Fr OspA 80 < 10 40 40 OspA + alum 80 < 10 80 80 OspA + QS-21 1,280 640 640 320 OspB 20 < 10 < 10 < 10 OspB + alum 320 < 10 < 10 < 10 OspB + QS-21 320 < 10 < 10 160 Normal mouse serum ≤ 10 ≤ 10 ≤ 10 ≤ 10 1 Borreliacidal activity of mouse antisera to OspA and OspB against different geographic isolates of B. burgdorferi was determined by me colorimetric assay of the present invention as described below. Serially diluted, heat- inactivated sera in mBSK containing 120 μg of phenol red per ml were incubated with spirochetes (approximately 4 x 10 6 of spirochetes) and complement source in 96- well microliter plates. After 48 to 96 h incubation at 32° C, the absorbance at 562/630 nm was measured using a microplate reader. The titer of borreliacidal activity was defined as the highest dilution of me antisera which inhibited absorbance change caused by 50% (2 x 10 6 bacteria) of spirochetes used in this assay. Further, humoral immune responses to experimental Lyme disease Osp subunit vaccines and to natural borrelial infection in dogs were investigated by characterization of immune sera and naturally exposed dog sera isolated in the New York area. The experimental subunit vaccines were formulated with adjuvant QS-21, OspA and/or OspB. Beagles were subcutaneously vaccinated twice at 12 and 16 weeks of age, and two weeks after the second vaccination immune sera was isolated for analysis of immunoglobulin (Ig) G isotype antibody responses and antiborrelial activity. QS-21 formulated vaccine containing liproproteins OspA and OspB elicited 4-fold higher IgGl (p<0.1) and 8-fold higher IgG2 (p<0.05) antibody responses than non-QS-21 formulated vaccines. Nonlipidated OspA and OspB based vaccine formulated with QS-21 elicited significantly (p < 0.005) lower IgGl and IgG2 antibody titer than lipoproteins OspA and OspB based vaccine. Antisera induced by the vaccine containing QS-21, OspA and OspB possessed high titer of antibodies to both proteins. Antisera induced by either OspA or OspB based vaccine had high titers of antibody and antiborrelial activity against B. burgdorferi sensu stricto strains B31 and CA-2-87 as those elicited by OspA and OspB based vaccine. However, only the latter were also antiborrelial to heterologous borrelial strain 24008 Fr and B. garinii sp. nov. strain G25, both European isolates. Antisera to vaccine formulated with lipoproteins OspA and OspB possessed significantly (p < 0.005) higher antiborrelial activity than those to vaccine formulated with nonlipidated OspA and OspB. Among 63 naturally exposed B. burgdorferi specific antibody positive dog sera, 24 (38%) sera had antibodies to both OspA and OspB; 3 (5%) and 4 (6%) sera had antibody to only OspA and OspB, respectively. Titers of OspA and OspB specific antibodies were low. Thirteen of 63 (21 %) sera had low titer of antiborrelial activity; 7 of these 13 sera had antibodies neither to OspA nor to OspB. Thus, (1) immunogenicity of OspA and OspB based subunit vaccine can be greatly enhanced using lipoproteins OspA and OspB; (2) immunogenicity of Osp subunit vaccine can be further enhanced by the adjuvant QS-21; (3) OspA and OspB based vaccine was more potent than a single protein based vaccine in eliciting functional humoral immune response in dogs. This study demonstrated that a QS-21 formulated, FLOspA and FLOspB based vaccine was able to elicit maximal humoral immune response in dogs. The enhanced immune response may have resulted from the double adjuvant activity of the lipid component of the full length outer surface proteins and QS-21. The lipid structure of OspA has been suggested to possess adjuvant activity and is required for enhanced immunogenicity of OspA in mice (Erdile et al, Infect. Immun. 67:81-90 (1993)). This is consistent with the present observation that in the formulation with QS-21, lipidated OspA and OspB were significantly (p< 0.005) more immunogenic than nonlipidated OspA and OspB, and induced significantly (p < 0.005) higher functional antibody response in dogs. As described above, QS-21 is superior to aluminum hydroxide in enhancing humoral immune response to truncated nonlipidated OspA and OspB in mice. Aluminum hydroxide is incapable of enhancing antibody response to FLOspA (Erdile et al, Infect. Immun. 67:81- 90 (1993)). However, the present data clearly demonstrated that QS-21 was also capable of significantly enhancing humoral immune response to FLOspA and FLOspB in dogs. Moreover, QS-21 induced broadening of isotype antibody responses in dogs as in mice, which will enhance host immunity by enhancing bactericidal and opsonophagocytic activity against spirochetal infection (Benach et al, J. Infect. Dis. 150:491-501 (1984); Kochi et al, Infect. Immun. 56:314-21 (1988), Peterson et al, Infect. Immun. 46:608-11 (1984). The present data showed that FLOspA and FLOspB based vaccine formulated with QS-21 was much more potent than either FLOspA or FLOspB based vaccine in inducing functional immune response. OspA or OspB based, QS-21 formulated vaccine was capable of inducing an antibody response which was antiborrelial against an homologous B. burgdorferi sensu stricto strain B31 and a heterologous strain CA-2-87 (a California isolate), but not against two European isolates strain 24008 Fr (a French isolate) and B. garinii sp. nov. strain G25 (a Swedish isolate). However, OspA and OspB based, QS-21 formulated vaccine elicited antibodies which were also antiborrelial to these two European isolates. This indicated that weakly immunogenic, antiborrelial epitope(s) may be conserved on OspA and OspB among these strains, and that antibodies to these epitopes may have had synergistic antiborrelial action on spirochetes. At least three genospecies and six different OspA serogroups of Lyme disease spirochetes have been recognized (Baranton et al, Int. J. Sys. Bacteriol. 42:378-83 (1992); Wilske et al, J. Clin. Microbiol 57:340-50 (1993)). Strains B31 and G25 have been designated as OspA serogroups 1 and 6, respectively (Wilske et al, J. Clin. Microbiol 57:340-50 (1993)). OspA and OspB of strains B31, CA-2-87, G25 have different electrophoresis profile (see Figures 19A-19C). Cross antiborrelial activity against different genospecies and OspA serogroups of spirochetes of the antisera elicited by the experimental vaccine showed promise for developing an effective vaccine against Lyme disease for different geographic regions. The high functional activity of antisera elicited by QS-21 formulated vaccine may have resulted from enhanced antibody titer and broadening antibody responses to functional epitopes on OspA and OspB of the spirochete. Antiborrelial activity of canine antisera appeared to be associated with isotype IgG2 antibody (R=0.61), not with IgGl antibody (R=0.13). This may be due to higher titer of IgG2 antibody responses elicited by the vaccine. It has been reported that dogs naturally infected by Lyme disease spirochete did not have detectable antibodies to OspA (Greene et al, J. Clin. Microbiol. 26:648-53 (1988)). In the present study, 63 naturally exposed canine sera have been identified which have specific antibodies to Lyme disease spirochete. Forty-nine percent of these sera had antibodies to OspA and/or OspB, which is inconsistent with the previous report (Greene et al, J. Clin. Microbiol 26:648-53 (1988)). The serum samples of the present invention were isolated from Westchester and Long Island, New York, a highly endemic region for Lyme disease (Alpert et al, NY State J. Med. 92:5- 8 (1992)). Repeat infection of dogs by Lyme disease spirochetes in this area may explain the different observations. Although relatively large number of sera samples contained OspA and OspB specific antibodies, their titers were low. It has been suggested that the quality of immune response to OspA and OspB is dependent on the antigen load (Schaible et al, Immunol. Let. 36:219- 26 (1993)). This poor antibody response to natural borrelial infection may be related to the quantity of spirochetes delivered by tick bite. In addition, only 13/63 (21 %) naturally exposed sera had detectable antiborrelial activity, and 7 of them did not have antibodies to OspA and OspB, indicating antibodies to other than OspA and OspB are also antiborrelial. These protective antigens may be candidates for vaccines. In summary, QS-21 formulated, lipoproteins OspA and OspB based experimental vaccines were able to elicit high humoral immune response in dogs regardless of the fact that the natural borrelial infection stimulates poor antibody responses to the protective immunogens. The immune sera were highly antiborrelial to both homologous and heterologous Lyme disease spirochetes. The present experimental vaccines have also conferred protection from spirochete infected tick challenge of dogs, thus showing potential for the development of subunit vaccines for dogs and humans against Lyme disease. Table 3. OspA and OspB specific antibody and antiborrelial activity of naturally exposed, seropositive dog sera. Immunoblot band Reaction Serum no. Antiborrelial titer OspA and OspB +" 6 10 to 160 OspA and OspB + 18 _b OspA and OspB - 7 10 to 80 OspA and OspB - 25 - OspA only + 3 - OspB only + 4 - + a : Positive; - : Negative. Immunoblotting was performed as described in Fig. 13. Antiborrelial activity of sera was determined according to me method of me present invention. Heat-inactivated, filter-sterilized sera were serially diluted in 100 μl of mBSK containing 120 μg of phenol red per ml in duplicate in 96- well plates. Five μl of sterile guinea pig complement and 95 μl of spirochete (containing approximately 4 x 10 6 of spirochetes) of strain B31 in fresh mBSK were added to each well. After 48 h incubation at 32°C, the absorbance at 562/630 nm was measured using a microplate reader. The titer of borreliacidal activity was defined as the highest dilution of the antisera which inhibits absorbance change caused by 50% (2 x 10 6 bacteria) of spirochetes used in the assay. The proteins OspA and OspB and fragments thereof may be obtained as described in the present specification or according to any other methods known in the literature. See, for example, Erdile, L. F. et al. , Infect. Immun. 67:81-90 (1993); Fikrig, E. et al , Science 250:553-556 (1990); Bergstrom, S. et al, Mol Microbiol. 5:479-86 (1989); Howe, T.R. et al, Science 227:645-6 (1985); Rosa, P. A. et al, Mol Micwbiol 6:3031-40 (1992); Simon, M.M et al, J. Infect. Dis. 164:123-32 (1991), the contents of which are fully incorporated by reference herein. The terms "OspA" or "OspB" as used herein include lipidated and non- lipidated as well as acylated and non-acylated forms of the outer surface proteins A and B, unless indicated otherwise. The term "saponin" as used herein includes glycosidic triterpenoid compounds which produce foam in aqueous solution, have hemolytic activity in most cases, and possess immune adjuvant activity. The invention encompasses the saponin per se, as well as natural and pharmaceutically acceptable salts and pharmaceutically acceptable derivatives. The term "saponin" also encompasses biologically active fragments thereof. Adjuvant saponins have been identified and purified from an aqueous extract of the bark of the South American tree, Quillaja saponaria Molina. See, U.S. Patent No. 5,057,540, the contents of which are fully incorporated by reference herein. At least 22 peaks with saponin activity were separable. The predominant purified Quillaja saponins have been identified as QS-7, QS- 17, QS-18, and QS-21. These saponins have been purified by high pressure liquid chromatography (HPLC) and low pressure silica chromatography. These four saponins have adjuvant effect in mice. QS-21 was further purified using hydrophilic interaction chromatography (HILIC) and resolved into two peaks, QS-21-V1 and QS-21-V2, which have been shown to be different compounds. These saponins are useful as immune adjuvants and enhance immune responses in individuals at a much lower concentration than the previously available heterogeneous saponin preparations without the toxic effects associated with crude saponin preparations. "QS-21 " designates the mixture of components QS-21-V1 and QS-21- V2 which appear as a single peak on reverse phase HPLC on Vydac C4 (5 μm particle size, 330 A pore, 4.6 mm ID x 25 cml) in 40 mM acetic acid in methanol/ water (58/42, v/v). The component fractions are referred to specifically as QS-21-V1 and QS-21 -V2 when describing experiments or results performed on the further purified components. In the preferred embodiment, the saponins of the present invention are purified from Quillaja saponaria Molina bark. Aqueous extracts of the Quillaja saponaria Molina bark were dialyzed against water. The dialyzed extract was lyophilized to dryness, extracted with methanol and the methanol- soluble extract was further fractionated on silica gel chromatography and by reverse phase high pressure liquid chromatography (RP-HPLC). The individual saponins were separated by reverse phase HPLC as described in Example 1. At least 22 peaks (denominated QS-1 to QS-22) were separable. Each peak corresponded to a carbohydrate peak and exhibited only a single band on reverse phase thin layer chromatography. The individual components were identified by retention time on a Vydac C 4 HPLC column as follows: Peak Retention Time (minutes) QS-1 solvent front QS-2 4.6 QS-3 5.6 QS-4 6.4 QS-5 7.2 QS-6 9.2 QS-7 9.6 QS-8 10.6 QS-9 13.0 QS-10 17.2 QS-11 19.0 QS-12 21.2 QS-13 22.6 QS-14 24.0 QS-15 25.6 QS-16 28.6 QS-17 35.2 QS-18 38.2 QS-19 43.6 QS-20 47.6 QS-21 51.6 QS-22 61.0 The purified saponins are characterized by carbohydrate content, reverse phase and normal phase TLC, UV, infra red, NMR spectra, and fast atom bombardment - mass spectroscopy. The approximate extinction coefficient determined for 1 % (w/v) solutions in methanol at 205 nm of several of the more preferred purified saponins are as follows: 1 % ^205 nm QS-7 34 QS-17 27 QS-18 27 QS-21 28 Carbohydrate content was used to quantitate the saponins in some instances. The carbohydrate assay was the anthrone method of Scott and Melvin (Anal. Chem. 25:1656 (1953)) using glucose as a standard. This assay was used to determine a ratio of extent of anthrone reaction (expressed in glucose equivalents) per mg of purified saponin (dry weight) so that dry weight of a particular preparation could be estimated by use of anthrone assay. It must be noted that differences in reactivity with anthrone for different saponins may be due to carbohydrate composition rather than quantity as different monosaccharides react variably in this assay. The substantially pure QS-7 saponin is characterized as having immune adjuvant activity, containing about 35% carbohydrate (as assayed by anthrone) per dry weight, having a uv absorption maxima of 205-210 nm, a retention time of approximately 9 - 10 minutes on RP-HPLC on a Vydac C 4 column having 5 μm particle size, 330 A pore, 4.6 mm ID x 25 cm L in a solvent of 40 mM acetic acid in methanol/water (58/42; v/v) at a flow rate of 1 ml/min, eluting with 52-53 % methanol from a Vydac C 4 column having 5 μm particle size, 330 A pore, 10 mM ID X 25 cm L in a solvent of 40 mM acetic acid with gradient elution from 50 to 80% methanol, having a critical micellar concentration of approximately .06% in water and .07% in phosphate buffered saline, causing no detectable hemolysis of sheep red blood cells at concentrations of 200 μg/ml or less, and containing the monosaccharide residues terminal rhamnose, terminal xylose, terminal glucose, terminal galactose, 3-xylose, 3,4-rhamnose, 2,3-fucose, and 2,3-glucuronic acid, and appose (linkage not determined). The substantially pure QS-17 saponin is characterized as having adjuvant activity, containing about 29% carbohydrate (as assayed by anthrone) per dry weight, having a UV absorption maxima of 205-210 nm, a retention time of approximately 35 minutes on RP-HPLC on a Vydac C 4 column having 5 μm particle size, 330 A pore, 4.6 mm ID x 25 cm L in a solvent of 40 mM acetic acid in methanol-water (58/42; v/v) at a flow rate of 1 ml/min, eluting with 63-64% methanol from a Vydac C 4 column having 5 μm particle size, 330 A pore, 10 mm ID x 25 cm L in a solvent of 40 mM acetic acid with gradient elution from 50 to 80% methanol, having a critical micellar concentration of .06% (w/v) in water and .03% (w/v) in phosphate buffered saline, causing hemolysis of sheep red blood cells at 25 μg/ml or greater, and containing the monosaccharide residues terminal rhamnose, terminal xylose, 2-fucose, 3-xylose, 3,4-rhamnose, 2,3-glucuronic acid, terminal glucose, 2-arabinose, terminal galactose and appose (linkage not determined). The substantially pure QS-18 saponin is characterized as having immune adjuvant activity, containing about 25-26% carbohydrate (as assayed by anthrone) per dry weight, having a UV absorption maxima of 205-210 nm, a retention time of approximately 38 minutes on RP-HPLC on a Vydac C 4 column having 5 μm particle size, 330 A pore, 4.6 mm ID x 25 cm L in a solvent of 40 mM acetic acid in methanol/water (58/42; v/v) at a flow rate of 1 ml/min, eluting with 64-65 % methanol from a Vydac C 4 column having 5 μm particle size, 330 A pore, 10 mm ID x 25 cm L in a solvent of 40 mM acetic acid with gradient elution from 50 to 80% methanol, having a critical micellar concentration of .04% (w/v) in water and .02% (w/v) in phosphate buffered saline, causing hemolysis of sheep red blood cells at concentrations of 25 μg/ml or greater, and containing the monosaccharides terminal arabinose, terminal appose, terminal xylose, terminal glucose, terminal galactose, 2-fucose, 3-xylose, 3,4-rhamnose, and 2,3-glucuronic acid. The substantially pure QS-21 saponin is characterized as having immune adjuvant activity, containing about 22% carbohydrate (as assayed by anthrone) per dry weight, having a UV absorption maxima of 205-210 nm, a retention time of approximately 51 minutes on RP-HPLC on a Vydac C 4 column having 5 μm particle size, 330 A pore, 4.6 mm ID x 25 cm L in a solvent of 40 mM acetic acid in methanol/water (58/42; v/v) at a flow rate of 1 ml/min, eluting with 69 to 70% methanol from a Vydac C 4 column having 5 μm particle size, 330 A pore, 10 mm x ID 25 cm L in a solvent of 40 mM acetic acid with gradient elution from 50 to 80% methanol, with a critical micellar concentration of about .03% (w/v) in water and .02% (w/v) in phosphate buffered saline, and causing hemolysis of sheep red blood cells at concentrations of 25 μg/ml or greater. The component fractions, substantially pure QS-21-V1 and QS-21-V2 saponins, have the same molecular weight and identical spectrums by FAB-MS. They differ only in that QS-21 - VI has a terminal appose which is xylose in QS-21 -V2 (which therefore has two terminal xyloses and no appose). The two components additionally contain the monosaccharides terminal arabinose, terminal appose, terminal xylose, 4-rhamnose, terminal galactose, 2-fucose, 3-xylose, and 2,3-gluc¬ uronic acid. As described in the present specification, QS-21 significantly enhances the immunogenicity of truncated OspA (TOspA) and OspB (TOspB) of B. burgdorferi sensu stricto strain B31 and broadens immunoglobulin (Ig) G antibody responses in mice. Further, the immunological properties of QS-21 formulated, OspA and/or OspB based experimental Lyme disease vaccines were characterized in dogs. The data showed that QS-21 significantly enhanced antibody response to the experimental vaccines. Vaccine elicited antisera had high titer of antiborrelial activity. Only the experimental vaccine containing both full length OspA (FLOspA) and OspB (FLOspB) induced antibodies which were antiborrelial not only to the homologous strain, but also to the heterologous and different genospecies of Lyme disease spirochetes. The vaccines of the invention are useful as vaccines which induce active immunity toward antigens in individuals. Preferably, such individuals are humans, however the invention is not intended to be so limiting. Any animal which may experience the beneficial effects of the vaccines of the invention are within the scope of animals which may be treated according to the claimed invention. The vaccines of the present invention induce active immunity when administered over a wide range of dosages and a wide range of ratios to the antigen being administered. In one embodiment, the saponin is administered in a ratio of adjuvant to OspA/OspB (w/w) of 3.0 or less, preferably 1.0 or less. The OspA, OspB and saponin may be administered either individually or admixed with other substantially pure adjuvants to achieve the enhancement of the immune response. Moreover, the vaccines of the present invention may comprise a single saponin or mixtures of saponins. The mixtures of the saponins may be purified saponins or crude mixtures of saponins. Among the saponin mixtures effective in the present invention are fractions QS-7 and QS-17, QS-7 and QS-18, QS-17 and QS-18, or QS-7, QS- 17, and QS-18 administered together. Purified saponins may also be administered together with non-saponin adjuvants. Such non-saponin adjuvants useful with the present invention are oil adjuvants (for example, Freund's Complete and Incomplete), liposomes, mineral salts (for example, AlK(SO 4 ) 2 , AlNa(SO 4 ) 2 , AlNH 4 (SO 4 ), silica, alum, Al(OH) 3 , Ca 3 (PO 4 ) 2 , kaolin, and carbon), polynucleotides (for example, poly IC and poly AU acids), and certain natural substances (for example, wax D from Mycobac- terium tuberculosis, as well as substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella), conjugates to the carrier proteins, such as bovine serum albumin, diphtheria toxoid, tetanus toxoid, edestin, keyhole-limpet hemocyanin, Pseudomonal Toxin A, choler- agenoid, cholera toxin, pertussis toxin, viral proteins, and eukaryotic proteins such as interferons, interleukins, or tumor necrosis factor. Such proteins may be obtained from natural or recombinant sources according to methods known to those skilled in the art. When obtained from recombinant sources, the non- saponin adjuvant may comprise a protein fragment comprising at least the immunogenic portion of the molecule. Other known immunogenic macromolecules which may be used in the practice of the invention include, but are not limited to, polysaccharides, tRNA, nonmetabolizable synthetic polymers such as polyvinylamine, polymethacrylic acid polyvinylpyrrolidone, mixed polycondensates (with relatively high molecular weight) of 4'4'- diaminodiphenyl-methane-3,3'-dicarboxylic acid and 4-nitro-2-aminobenzoic acid (See Sela, M., Science 766:1365-1374 (1969)) or glycolipids, lipids or carbohydrates. A preferable adjuvant is alum, which gives a 3-fold increase in IgGl response. The saponins may also be directly linked to the antigen or may be linked via a linking group. By the term "linker group" is intended one or more bifunctional molecules which can be used to covalently couple the saponin or saponin mixture to the OspA and OspB proteins and which do not interfere with the production of antigen-specific antibodies in vivo. The linker group may be attached to any part of the saponin so long as the point of attachment does not interfere with the production of antigen-specific antibodies in vivo and thus interfere with the induction of active immunity. Examples of linker groups which can be used to link the saponin to the OspA and OspB proteins may comprise -NH- (CH2) q -NH- , wherein q i s 2-10 ; -0- (CH 2 ) r -NH- , wherei n r i s 2-10 ; 0 0 -X- (CH 2 ) s -X-(!- (CH2)t - . wherein X = NH , S or 0. s = 2-5 , t = 2-12 ; 0 0 -C- (CH ) U C- , wherein u = 2-12; 0 -Y-(CH 2 ) V -C- . wherein Y i s NH or S , v = 1-3. Typically, the saponins are linked to the OspA and OspB proteins by the preparation of an active ester of glucuronic acid, a component of the saponins, followed by reaction of the active ester with a nucleophilic functional group on the protein. Examples of the active esters which may be used in the practice of the invention include me glucuronate of N- hydroxysuccinimide, sulfo-N-hydroxysuccinimide, hydroxybenzotriazole, and p-nitrophenol. The active esters may be prepared by reaction of the carboxy group of the saponin with an alcohol in the presence of a dehydration agent such as dicyclohexylcarbodiimide (DCC), l-(3-dimethylaminopropyl)-3- ethylcarbodiimide (EDC) , and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide (EDCI). The use of EDC to form conjugates is disclosed in U.S. Patent No. 4,526,714 to Feijen et al. and PCT application publication no. WO91/01750, and Arnon, R. et al , Proc. Natl. Acad. Sci. (USA) 77:6769- 6772 (1980), the disclosures of which are fully incorporated by reference herein. The protein is then mixed with the activated ester in aqueous solution to give the conjugate. Where a linker group between the saponin and the protein is desired, the active ester of the saponin glucuronate is prepared as described above and reacted with the linker group, e.g. 2-aminoethanol, an alkylene diamine, an amino acid such as glycine, or a carboxy-protected amino acid such as glycine rt-butyl ester. If the linker contains a protected carboxy group, the protecting group is removed and the active ester of the linker is prepared (as described above). The active ester is then reacted with the antigen to give the conjugate. Alternatively, the antigen may be derivatized with succinic anhydride to give an antigen-succinate conjugate which may be condensed in the presence of EDC or EDCI with a saponin-linker derivative having a free amino or hydroxyl group on the linker. See WO91/01750. Once derivatized at the glucuronate carboxy 1 with a linker group, the saponins retain adjuvant activity. Those saponin derivatives prepared by reductive alkylation at the triterpene aldehyde do not appear to retain adjuvant activity at doses less than 40 μg. However, derivatives in which the saponin triterpene aldehyde was reduced to an alcohol by sodium borohydride reduction did retain some activity. It is also possible to prepare a saponin conjugate comprising a linker with a free amino group (derived from an alkylene diamine) and crosslink the free amino group with a heterobifunctional cross linker such as sulf osuccinimidyl 4-(N-maleimidocyclohexane)- 1 -carboxy late which will react with the free sulfhydryl groups of protein antigens. The saponin may also be coupled to a linker group by reaction of the aldehyde group of the quillaic acid residue with an amino linker to form an intermediate imine conjugate, followed by reduction with sodium borohydride or sodium cyanoborohydride. Examples of such linkers include amino alcohols such as 2-aminoethanol and diamino compounds such as ethylenediamine, 1 ,2-propylenediamine, 1 ,5-pentanediamine, 1 ,6- hexanediamine, and the like. The antigen may then be coupled to the linker by first forming the succinated derivative with succinic anhydride followed by condensation with the saponin-linker conjugate with DCC, EDC or EDCI. In addition, the saponin may be oxidized with periodate and the dialdehyde produced therefrom condensed with an amino alcohol or diamino compound listed above. The free hydroxyl or amino group on the linker may then be condensed with the succinate derivative of the protein in the presence of DCC, EDC or EDCI. The ratio of saponin molecules per protein molecule may vary considerably according to the molecular weight of the antigen, the number of binding sites on the protein capable of being coupled to the saponin, and the antigenic characteristics of the particular saponin. In general, the ratio of saponin molecules to protein molecules may be about 0.1:1 to about 10: 1. Preferably, the ratio may range from about 1:1 to about 3:1. Administration of the vaccines of present invention may be by parenteral, intravenous, intramuscular, subcutaneous, intranasal, or any other suitable means. The dosage administered may be dependent upon the age, weight, kind of concurrent treatment, if any, and nature of the antigen administered. In general, the vaccines may be administered at a dosage of about 0.01 to about 1.0 mg/kg of protein and saponin per weight of the individual. The initial dose may be followed up with a booster dosage after a period of about four weeks to enhance the immunogenic response. Further booster dosages may also be administered. The effective compound useful in the method of the present invention may be employed in such forms as capsules, liquid solutions, suspensions or elixirs for oral administration, or sterile liquid forms such as solutions or suspensions. Any inert carrier is preferably used, such as saline, or phosphate-buffered saline, or any such carrier in which the compounds used in the method of the present invention have suitable solubility properties for use in the methods of the present invention. The vaccines of the present invention may also be encapsulated within liposomes according to U.S. Patent No. 4,235,877 to Fullerton. The invention also provides for a kit for the immunization of an animal comprising a carrier compartmentalized to receive in close confinement therein one or more container means wherein a first container contains the OspA and OpsB proteins and, optionally, the saponin. The kit may instead include at least one other container means which contain a saponin adjuvant or other adjuvant as described herein. The invention also relates to a method of inducing immunity to B. burgdorferi in an animal, comprising administering to the animal a vaccine comprising OspA, OspB or fragments thereof; and a saponin adjuvant. Having now generally described the invention, the same may be further understood by reference to the following examples, which are not intended to be limiting unless so expressly stated. Example 1 A Colorimetric Microtiter Assay for Borreliacidal Activity of Antisera Materials and Methods Bacterial Strains and Growth Conditions Borrelia burgdorferi strains B31 (Burgdorfer, W., et al , Science 267:1317-1319 (1982)) and Fr isolated from ticks in Germany were kindly provided by H.-J. Wellensieki, Klinikum Der Justus-Liebig-Universitat Giessen, Germany. Strains G25 (Postic, D., et al , Res. Microbiol. 141:465- 475 (1990)) and Cr, isolated from ticks in Sweden and Wisconsin, USA, were generously provided by A. Barbour, University of Texas Health Science Center, San Antonio, TX, and S. Callister, Gundersen Medical Foundation, La Crosse, WI, respectively. Spirochetes were grown in mBSK medium at 32°C as described previously (Callister, S.M., et al , J. Clin. Microbiol. 25:363-365 (1990)). Antisera and Normal Sera Antiserum to lysates of B. burgdorferi was raised by subcutaneous immunization of C3H/HeJ mice at age of 6 to 7 weeks. One hundred μl of the spirochete lysates containing 25 μg of protein in saline was mixed with an equal volume of Freund's Complete Adjuvant for the first immunization. The same dose of the antigen was mixed with an equal volume of Freund's Incomplete Adjuvant, and given for the next two immunizations at two week intervals. Antiserum to a recombinant outer surface protein A derived from strain B31 (OspA-B31) (Hung, C.-H, et al , "Expression, purification and characterization of outer surface protein A (OspA) and B (OspB) from the Lyme disease spirochete, Borrelia burgdorferi," V Int. Con Lyme Borreliosis, Arlington, VA, USA (1992)) was produced by subcutaneous immunization of the mice 3 times with 200 μl of the antigen containing 25 μg of the recombinant protein and 20 μg of saponin adjuvant (QS-21) (Kensil, C.R., et al , J. Immunol 146:431-431 (1991)) at two week intervals. Antiserum was isolated two weeks after the last immunization. Normal guinea pig complement (276 C'H 50 /ml) (GIBCO, Grand Island, NY) was stored at -80°C. The complemented was filter-sterilized and used immediately after thawing. Colorimetric Borreliacidal Assay This CBA was performed in 96-well plate (Costar, Cambridge, MA). The appropriate concentration for each reagent used in the CBA was first determined by checkerboard titration. Each serum sample was prepared by mixing an equal volume of sera from at least ten mice, inactivated by heating at 56 °C for 45 min and sterilized by filtration through a 0.45 μm centrifuge filter (Spin-X, Costar, Cambridge, MA). Each sample was tested in triplicate. The sera were serially diluted in 100 μl of mBSK containing 120 μg/ml phenol red (SIGMA, St. Louis, MO) (mBSK-PR). Five μl of guinea pig complement were added to each well, and mixed. The spirochete culture in mBSK at the logarithmic growth phase was centrifuged for 8 min at 9000 x g and resuspended in fresh mBSK medium. Ninety-five μl of spirochete suspension (containing approximately 4 x 10 6 spirochetes) were added to each well (final volume 200 μl). After mixing, the plates were incubated at 32 °C for 2 to 5 days. The absorbance at 562/630 nm of each well was measured using a microplate reader (Bio-tek Instruments, Burlington, VT) before and every 24 h after incubation. For some assays, 5 μl of mBSK were applied instead of guinea pig complement. Controls consisting of mBSK instead of serum with or without complement were also included in each assay. For evaluation of killing percentage, the absorbance at 562/630 nm of the bacterial growth controls containing different concentration of spirochetes in mBSK was also determined by incubation of 100 μl of mBSK-PR with 100 μl of the serially diluted spirochetes in mBSK at 32 °C. Radioactive fHJ-Thymidine Incorporation Assay The [ 3 H]-thymidine uptake by B. burgdorferi was performed in triplicate in 96- well plates. One hundred μl of borrelial suspension (approximately 8 x 10 6 spirochetes) was serially (two-fold) diluted in mBSK, and incubated with an equal volume of mBSK-PR at 32°C. After 30 h incubation, 20 μl of pHJ-thymidine in mBSK (100 μCi/ml) were added into each well. After another 18 h of incubation for pulse-labeling the live spirochetes (Pavia, C.S., et al , J. Infect. Dis. 163:656-659 (1991)), the absorbance at 562/630 of the plate was measured by a microplate reader, and the radioactivity was determined. Samples were harvested onto glass-fiber filter and washed 20 times using a PHD cell harvester (Cambridge Technology, Inc., Cambridge, MA). Each filter disk retaining spirochetes was transferred into a scintillation vial containing scintillation cocktail (CytoScint, ICN Biomedicals, Inc., Irvine, CA), and the radioactivity measured as counts per minutes using liquid scintillation system LS 1701 (Beckman Instruments, Inc., Irvine, CA). Results The present CBA is based upon color changes that occur resulting from the accumulation of nonvolatile acid produced by actively metabolizing spirochetes after incubation of a certain period of time in the presence of phenol red. The color changes due to the accumulation of the acid could be detected qualitatively by the naked eye or quantitatively by a microplate reader. A number of factors, such as concentration of phenol red, wavelength used for measuring the absorption, spirochete concentration, and buffering capacity of the growth medium could affect the CBA. Solutions containing different concentrations of phenol red (2.3 μg/ml to 133 μg/ml) were first titrated with HC1 (final concentration of 0.77 mM to 9.6 mM), and die absorbance was determined using various single wavelengths (405 nm, 450 nm, 490 nm, 562 nm, 595 nm, and 630 nm) or dual wavelengths (405/630, 450/630, 490/562, 562/630). The concentration of phenol red (final concentration 60 μg/ml) and dual wavelength (562/630 nm) were chosen because they reflected the maximal absorbance change. A proper concentration of spirochetes used in this assay was also tested. With low concentrations of the spirochetes, the color or absorbance change of the assay mixture was delayed or minimized due to the inadequate accumulation of acids. While with high concentrations of spirochetes, more specific antibodies would be required to kill the bacteria and inhibit the color change. Thus, an optimal concentration of 4 x 10 6 spirochetes was chosen to ensure the sensitivity of this assay. A yellow color of the assay mixture after incubation for 48 to 72 h indicates growth of Borrelia, whereas a red color represents either no borrelial growth or borrelial death (Fig. 1). To confirm the validity and reliability of the colorimetric assay, both [ 3 H]-thymidine uptake by live spirochetes and colorimetric changes due to borrelial growth were determined simultaneously with different concentrations of the bacteria. The [ 3 H]-thymidine incorporation by spirochetes or changes of the absorbance were dose-dependent (Fig. 2). The results of TIA correlated well with those of the colorimetric assay (R= 0.977), and were linearly related from an absorbance of 0.4 to 1.3 (Fig. 2). The CBA of mouse antisera to OspA-B31 is shown in Fig. 3. At nearly all serum dilutions, the absorbance readings increased from the 0 to 24 h time points. However, by 48 h after incubation, the absorbance readings decreased, and continued to decrease in a regular fashion through 120 h at all serum concentrations tested. With each of the first three dilutions (1: 10 to 1:40), the absorbance decreased approximately 0.1 optical density every 24 h. The absorbance decreased more dramatically as antisera were diluted further. Mouse antisera to strain B31 lysates strongly inhibited the growth of the homologous strain B31 after 48 h, as indicated by the high absorbance. However, normal mouse sera did not affect the growth of spirochete (Fig. 4), and was similar to borrelial growth controls in mBSK. The borreliacidal TIA and CBA using serially diluted antisera to B31 lysates were also done simultaneously. These two assays correlated well (R=0.935). The TIA and CBA also showed that the heat-inactivated mouse antisera in the presence or absence of guinea pig complement could equally and efficiently inhibit the growth of the spirochetes. Dark-field microscopy indicated that the number of live spirochetes were significantly reduced, and dead spirochetes were visible in the presence of the antisera, but not in the presence of the normal mouse sera. Example 2 Immunization of Animals with OspA, OspB and QS-21 The genes encoding strain B31 OspA or OspB were cloned using the published DNA sequences (Bergstrom, S. et al, Mol Microbiol. 5:479-486 (1989), the contents of which are fully incorporated by reference herein) and standard molecular biological techniques (Maniatis, T. et al, Molecular Cloning. Cold Spring Harbor Laboratory, New York (1982). Recombinant OspA starting at amino acid 17 and OspB starting at amino acid 20 were expressed in E. coli using a heat-inducible pL promotor (Mariciani, D.J. Vaccine 9:89-96 (1991); Nagi, K. et al, Nature 509:810-813 (1984)). In addition, each recombinant protein contained 16 amino acids (MVRABKRBEALRIAGS) (SEQ ID NO: 1) at its N-terminus derived from a bacterial leader and nucleotide linker sequence. Expression of OspA and OspB was confirmed with specific monoclonal antibodies to OspA and OspB of strain B31 (Cambridge Biotech, Corp., Worcester, Mass). OspA and OspB were purified by reversible citraconylation and anion exchange chromatography as described previously (Marciani, D.J. et al, in R. Burgess (ed.), protein purification, Alan R. Liss, Inc., New York, pp.443-458. ; Marciani, D.J. et al, Vaccine 9:89-96 (1991)). Antisera were prepared by subcutaneous immunization of 10 month old Beagles twice at two week interval with one ml of the Lyme vaccine. The vaccine contains 100 μg of truncated OspA and/or 100 μg of truncated OspB formulated with 50 μg of adjuvant QS-21. Antisera were isolated two weeks after second immunization. Borreliacidal activity of the sera was determined by CBA. Borrelial culture at logarithmic growth phase was centrifuged for 8 min at 9,000 xg at 15 °C and resuspended in fresh mBSK. Ninety five μl of the borrelial suspension (containing approximately 4x10 s of spirochetes) and 5 μl of guinea pig complement were mixed with an equal volume of the serially diluted heat-inactivated antisera in mBSK containing 120 μg of phenol red in microtiter plate, and incubated for 72 h at 32 °C. Absorbance at 562/630 was measured by a microtiter reader. The results are shown in Figure 5. High absorbance indicated borrelial death and high borreliacidal activity of the antisera. The low absorbance represented borrelial survival and growth. The error bars represent the standard error of 16 measurements of 8 serum samples for antisera and 8 measurements of 4 preimmune sera. Figure 6 shows the borreliacidal activity of canine antisera to truncated OspA and/ or OspB derived from B. burgdorferi strain B31 against the heterologous California strain CA-2-87. Sera preparation and borreliacidal assay were described as above except that the incubation time of borreliacidal assay was 96 h. High absorbance indicated borrelial death and high borreliacidal activity of the antisera. The low absorbance represented borrelial survival and growth. The error bars represent the standard error of 16 measurements of 8 serum samples for antisera and 8 measurements of 4 preimmune sera. Figure 7 shows the borreliacidal activity of individual canine antisera to truncated OspA and/or OspB derived from B. burgdorferi strain B31 against the homologous strain. Sera preparation and borreliacidal assay were described as above. This figure showed borreliacidal activity of individual dog antiserum and preimmune serum at 1:20 dilution. High absorbance indicated borrelial death and high borreliacidal activity of the antisera. The low absorbance represented borrelial survival and growth. Figure 8 shows the borreliacidal activity of individual canine antisera to truncated OspA and/or OspB derived from B. burgdorferi strain B31 against the heterologous California strain CA-2-87. Sera preparation and borreliacidal assay were described above. The figure shows borreliacidal activity of individual dog antiserum and preimmune serum at 1:80 dilution. High absorbance indicated borrelial death and high borreliacidal activity of the antisera. The low absorbance represented borrelial survival and growth. Figure 9 shows the borreliacidal activity of C3H/HeJ female mice that had been immunized twice with either 25 μg of truncated OspA, 25 μg of truncated OspB, or 25 μg of truncated OspA and 25 μg of truncated OspB. Sera from ten mice in each group were pooled and tested for borreliacidal activity against the homologous strain B31 or the highly divergent Swedish strain G25, as described above. Figure 10 shows the borreliacidal activity of C3H/Hej female mice that had been immunized twice with either 25 μg of truncated OspA + 20 μg of QS21 , 25 μg of truncated OspB + 20 μg of QS21 , or 25 μ of truncated OspA and 25 μg of truncated OspB + 20 μg of QS21. Sera from ten mice in each group were pooled and tested for borreliacidal activity against the homologous strain B31 or the highly divergent Swedish strain G25, as described above. Surprisingly, in dogs but not in mice, the results show that a combination of OspA and OpsB, when administered together with QS-21, results in a much greater borreliacidal activity when compared to when OspA was administered alone (Figures 5-9). Example 3 Characterization of Canine humoral Immune Responses to Osp Subunit Vaccines and to Natural Infection by Lyme Disease Spirochetes Materials and Methods Bacterial strains and growth conditions The bacterial strains were described above, in the corresponding section of Example 1 of the present specification. Borrelia burgdorferi sensu stricto strains B31, CA-2-87, and B. garinii sp. nov. strain G25 were also described elsewhere (Baranton et al, Int. J. Sys. Bacteriol. 42:378-83 (1992); Ma et al, J. Microbiol. Methods 77:145-53 (1993); Marconi et al, Infect. Immun. 67:2611-7 (1993)), the entire texts of which are herein incorporated by reference. Strain 24008 Fr, a French isolate, was generously provided by David Dennis (Center for Disease Control, Collins, CO). Spirochetes were grown in modified Barbour-Stoenner-Kelly (mBSK) medium at 32 °C as previously described (Callister, S. M. et al , J. Clin. Microbiol 25:363-386 (1990)), the entire text of which is herein incorporated by reference. More specifically, E. coli, strain MZ-1 was used as the host strain for expression of recombinant antigens. This strain was a gift from Takis Papas (National Cancer Institute, December 1984) and has the following genotype: galK ^ Δ BamN 7 M 53 CI857 Δ HI, his " , ilv, bio " , N + . (Nagai & Thogersen, Nature 509:810 (1984)). The expression vector was used to transfect MZ-1 strain cells with replication deficient bacteriophage lambda DNA sequences containing a temperature sensitive C7S57 transcription repressor gene. Under permissive temperature conditions, 32 °C, this repressor protein is active, inhibiting the synthesis of recombinant proteins. By shifting the cell culture to a nonpermissive temperature, 42°C, protein synthesis is induced. Use of this temperature sensitive mutant allows dense cell cultures to be achieved prior to the synthesis of recombinant proteins which may be toxic to the E. coli. The plasmids for expression of OspA and OspB were each transfected into host cell line MZ-1 which were then used for induction of recombinant antigens. Production of OspA and OspB OspA and OspB were produced according to Example 4 of the present specification and as further described below. The genes encoding OspA and OspB of B. burgdorferi sensu stricto strain B31 were cloned using published DNA sequence (Bergstrom, S. et al , Mol. Microbiol. 5:479-486 (1989)) and standard molecular biological techniques (Maniatis, T. et al, Molecular Cloning, Cold Spring Harbor Laboratory, New York (1982)). Recombinant OspA starting at amino acid 17 and OspB starting at amino acid 20 were expressed in E. coli strain MZ-1 using expression vector pLCBCl (Beltz, G. A. et al, U.S. Patent No. 4,753,873 (1988)), a vector very similar to that described by Lautenberger (Lautenberger, J. A. et al , Gene Anal. Techn. 7:63-66 (1984)) and used to express recombinant proteins to high level (Lautenberger, J. A. et al , Gene 25:75-84 (1983)). The vector makes use of the heat inducible pL promoter; and Shine-Delgarno sequence and first N-terminal 16 amino acid codons are derived from the Lambda ell gene. Cloning sites within three codons of mature OspA and OspB were chosen for ligation into the expression vector. The cloned OspA and OspB genes were confirmed by DNA sequencing (Bergstrom, S. et al. , Mol. Microbiol. 5:479- 486 (1989)). Expression of OspA and OspB was confirmed with specific monoclonal antibodies to OspA and OspB of strain B31 and polyclonal antiserum to the lysates of strain B31 (Ma, J. et al , Program and abstracts of V Int. Conf. Lyme Borreliosis, Arlington, Virginia (1992)). OspA and OspB were purified by reversible citraconylation and/or anion exchange hromatography as described previously (Marciani, D. J. et al , Vaccine 9:89- 96 (1991); Marciani, D. J. et al , in R. Burgess, ed. , Protein Purification, Alan R. Lise, Inc. , New York (1987) pp. 443-458). The truncated OspA and OspB started at amino acid 17 and 20, respectively. Recombinant proteins were expressed in Escherichia coli, confirmed, and purified as previously described (Marconi et al, Infect. Immun. 61:2611-1 (1993)), the entire text of which is herein incorporated by reference. More specifically, the expression plasmids pCBC10spA8+6 and pCBC10spB8+4, used to produce full length lipidated OspA and OspB proteins, were constructed and tested as follows: A. OspA and OspB Coding Sequences B. burgdorferi strain B31 (Burgdorfer et al , Science 267: 1317-1319 (1982)) was received from H.J. Wellenski, Klinikum Der Justus-Liebig- Universitat Giessen, Giessen, Germany. Total DNA extracted from this strain was used to subclone the OspA and OspB protein encoding regions separately. Figure 21 details each protein encoding region as they occur in B. burgdorferi strain B31. B. Expression Vector Construction The expression vector used to express B. burgdorferi recombinant OspA and OspB antigens was constructed by Cambridge Biotech Corporation (CBC) scientists and is based on published work (Shimatake & Rosenberg, Nature 297:128 (1981); Lautenberger et al , Gene 23:15 (1983); Lautenberger et al , Gene Analysis Techniques 7:63 (1984)). The vector uses the bacteriophage lambda pL promoter, and a fragment of the lambda ell gene which provides a ribosome binding site, ATG initiation codon, and 12 additional codons. A BamHI site follows which is used for cloning DNA fragments to be expressed, which in turn is followed by a polytranslation terminator. The basic features are presented in Figure 22. Commercially obtained lambda and pBR322 DNA were used as starting materials. C. Cloning of OspA and OspB for Expression 1. The OspA BamHI-digested fragment was subcloned into The Cambridge Biotech expression vector, pLCBCl, also restricted with BamHI. Plasmids from colonies that contained die 907 base pair insert were also digested with EcoRI (located distally to die 5' end) to determine me orientation of the fragment within pLCBCl . The correctly oriented clone was named pLCBC10spA8+6. Figure 23 details die entire sequence of pLCBC10spA8+6 and die origin of each base. 2. The OspB BamHI-restricted fragment was also subcloned (separately) into pLCBCl restricted widi BamHI. Plasmids from colonies that contained die 945 base pair fragment when digested with BamHI were also digested with Hindlll to determine the direction of the insertion. The clone wi i the correct orientation was named pLCBC10spB8+4. Figure 24 details the entire sequence of pLCBC10spB8+4 and shows die origin of each base. Table 4. Validation of the OspA Clone A series of restriction enzyme digests confirming pLCBC10spA8+6 were performed. The size of the DNA fragments expected is also presented. RESTRICTION MAPPING OF PLCB1OSPA8+6 (OSPA) Restriction Digests Predicted Fragment Sizes (bp) BamHI 3852, 902 PvuII 4754 EcoRI 3354, 1400 Hindlll 4754 Ndel 2766, 1988 Pstl 2612, 2142 Seal 3265, 1489 EcoRI + PvuII 3354, 880, 520 EcoRI + Hindπi 3354, 930, 470 Pstl + PvuII 2612, 1268, 874 The size of restriction fragments observed on the gel were indistinguishable from those predicted. See Figure 25. A deposit of the expression vector pLCBC10spA8+6 was made in compliance with the provisions of the Budapest Treaty, at American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, USA, on February 25, 1994, under accession number 69577. Table 5. Validation of the OspB Clone A series of restriction enzyme digests confirming pLCBC10spB8+4 were performed. The size of the DNA fragments expected is also presented. RESTRICTION MAPPING OF PLCBC1OSPB8+4 (OSPB) Restriction Digests Predicted Fragment Sizes (bp) BamHI 3852, 900 EcoRI 4752 Ndel 2766, 1986 Pstl 2562, 2190 PvuII 3700, 1052 BamHI + EcoRI 3105, 900, 747 EcoRI + Hindlll 3429, 1323 EcoRI + Pstl 2562, 1442, 748 Hindlll + Sspl 3240, 1512 PvuII + Sspl 2991, 1052, 709 The size of restriction fragments observed on the gel were indistinguishable from those predicted. See Figure 26. A deposit of the expression vector pLCBC10spB8+4 was made in compliance with the provisions of the Budapest Treaty, at American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, USA, on February 25, 1994, under accession number 69578. Monoclonal antibody Murine monoclonal antibody (MAb) to OspA and OspB of B. burgdorferi sensu stricto strain B31 were produced widi modification of previously described procedures (Lane, R.D., J. Immunol. Methods 57:223-8 (1985); Kennett, R.H., "Hybridomas: A New Dimension in Biological Analysis," in Monoclonal Antibodies, Kennett et al , Plenum Publ. Corp. , New York (1980), pp. 365-7). Briefly, Balb/c mice were immunized at least two times with recombinant OspA, OspB, or borrelial lysate. Primed spleen cells were isolated and fused witii the myeloma cell line SP2/0 by mixing at a 4:1 ratio in the presence of 50% polyethyleneglycol 1500 (Boehringer Mannheim, Indianapolis, IN). Hybridoma cultures producing antibodies of interest were cloned at least twice by limiting dilution. MAbs to OspA and OspB were identified by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Antibody isotypes were determined by ELISA using isotype- specific goat anti-mouse IgG conjugated to horseradish peroxidase (Pel-Freez, Rogers, AZ). MAbs were purified from culture supernatant using protein- A affinity column, and protein concentration determined by BCA assay (Pierce, Rockford, IL). Intrinsic radiolabeling of OspA and OspB and immunoprecipitation Escherichia coli strain MZ-1 harboring ospA or ospB gene was grown in 5 ml of LB broth to log phase at 32°C. Twenty μl of [9,10- 3 H]palmitic acid (52.4 Ci/mmol, Dupont Nen, Boston, MA) were added to the culture and incubated for another 2 h at 42 °C for heat induction of recombinant proteins. Bacteria were washed twice with cold sterile water, lysed with the solution of 0.05 Tris-HCl (pH 6.8), 1% Triton X-100, 0.5% sodium deoxycholate, 0.15% SDS, 0.15 M NaCl (Katona et al, Infect. Immun. 60:4995-5003 (1992)), and boiled for 10 minutes. The supernatant was incubated witii mixture of purified OspA specific MAbs H3G4, L3B5, T1F6, and T2H12, and with OspB specific MAb P4D1, respectively, for 2 h at 22 °C with gentle agitation, and ti en incubated widi protein G-Sepharose 4FF (Pharmacia, Piscataway, NJ) in 0.25 M Tris-HCl, pH 6.8, for another 1 h at 22°C. The mixture was microcentrifuged and washed 3 times, and resuspended in distilled water. The isolated recombinant proteins were mixed with sample buffer (Laemmli, U.K. , Nature (London) 227:680-5 (1970)), boiled for 5 min, and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Vaccine formulation Vaccine formulation was prepared as described in Example 4, below; also see Kensil et al, J. Immunol 746:431-7 (1990). Each dose (1 ml) of vaccine contained 100 or 25 μg each of FLOspA and/or FLOspB formulated with or without 50 μg of QS-21 (Kensil et al, J. Immunol. 146:431-1 (1990), in saline, pH 6.5. A vaccine containing 100 μg each of TOspA, TOspB and 50 μg of QS-21 was also prepared for comparative study. Vaccination and antiserum Beagle dogs (Harlan Sprague Dawley, Indianapolis, IN) at age of 12 weeks were vaccinated subcutaneously with 1 ml of vaccine. Immune response was boosted once 4 weeks later with the same dose of vaccine. Antiserum was isolated 2 weeks after the second vaccination and stored at -20°C. Naturally exposed canine sera Serum samples isolated from naturally exposed dogs in Westchester County and Long Island, New York, a highly endemic area for Lyme disease (Alpert et al, NY State J. Med. 92:5-8 (1992)) were kindly provided by H. Schneider, Vet Research, Farming Dale, New York, and Durland Fish, New York Medical College, Valhalla, New York. Those serum donors were not vaccinated with Ft. Dodge Lyme disease bacterin. Normal dog sera used as negative control were isolated from dogs raised in closed kennels (Harlan Sprague Dawley, Indianapolis, IN). SDS-PAGE and immunoblotting SDS-PAGE was performed using a 11 % separating gel (Edelman, R. , Vaccine 9:531-2 (1991)). Immunoblotting was conducted by modification of the procedure of Towbin et al. (Towbin et al, Proc. Natl Acad. Sci. USA 76:4350-4 (1979)). For testing canine serum samples, sera were diluted at 1:50, and immunoblot strips were used according to the instruction of manufacturer (Cambridge Biotech Corporation, Worcester, MA). The goat anti-dog IgG F(ab') 2 conjugated onto horseradish peroxidase (Bethyl Laboratories, Inc. , Montgomery, TX) were used to detect the specific antibody in serum samples. ELISA ELISA was performed using plates coated widi B. burgdorferi antigens, 0.2 μg OspA per well , and 0.2 μg OspB per well, respectively, as described (Lindenmayer et al, J.Clin. Microbiol 28:92-6 (1990)). Antibody isotypes were determined using isotype-specific goat anti-dog IgG conjugated to horseradish peroxidase (Bethyl Laboratories, Inc., Montgomery, TX). Antiborrelial assay A colorimetric microtiter assay for antiborrelial activity of antisera was performed as described above. Briefly, heat-inactivated, filter-sterilized sera were serially diluted in 100 μl of mBSK containing 120 μg of phenol red per ml in 96-well plates. Five μl of sterile guinea pig complement (Gibco BRL, Gaithersburg, MD) and 95 μl of spirochete (containing approximately 6 x 10 7 of spirochetes) in fresh mBSK were added to each well. After 48 to 72 h incubation at 32 °C, the absorbance at 562/630 nm was measured using a microplate reader. Antiborrelial activity was expressed by either absorbance change compared with those of preimmune sera or by titer. The titer of antiborrelial activity was defined as me highest dilution of the antisera which inhibited absorbance change caused by 50% of spirochetes used in this assay. Antiborrelial activity reflects borrelial killing and borrelial growdi inhibition by specific antibodies. Statistical analysis One-tailed Student's t-test was conducted to compare antibody titers of various groups of immune sera. Significance level was assigned assuming equal variances about the means using Microsoft Excel 4.0 for Macintosh. Results MAbs to OspA and OspB A number of MAbs reactive with OspA and OspB of B. burgdorferi sensu stricto strain B31 were developed. MAbs H3G11, L3B5, T1F6, T2H12 (all IgGl) are specific for OspA, and MAb P4D1 (IgGl) is specific for OspB, as tested by ELISA and immunoblotting. Radioimmunoprecipitation of OspA and OspB MAb P4D1 specific for OspB precipitated OspB from lysate of E. coli labeled with [9,10- 3 H]palmitic acid. A mixture of 4 OspA specific MAbs described above were used for precipitation of OspA. Lipidation of the precipitated OspA and OspB was analyzed by SDS-PAGE and fluorography. both TOspA and TOspB are nonlipidated proteins, and FLOspA and FLOspB are lipoproteins as shown by me radiolabeled bands (Fig. 11). FLOspB had another radiolabeled band with a molecular mass of approximately 22 KDa, perhaps a recombinant fragment from OspB (see Example 4, below). Antibody responses to experimental Osp vaccines Various experimental vaccines were evaluated in dogs for eliciting IgG isotype antibody responses (Fig. 12). A QS-21 formulated vaccine containing 100 μg each of FLOspA and FLOspB induced 30-fold higher (p < 0.005) IgGl and 4-fold higher (p < 0.0005) IgG2 antibody responses than die same formulation containing TOspA and TOspB. A vaccine containing 25 μg each of FLOspA and FLOspB was similar to the vaccine containing 100 μg each of the lipoproteins in terms of eliciting antibody titer. QS-21 enhanced at least 4-fold higher (p<0.1) IgGl and 8-fold higher (p <0.05) IgG2 antibody responses when comparing FLOspA and FLOspB based vaccines formulated witii and widiout QS-21. Either FLOspA and FLOspB based vaccine formulated wid QS-21 elicited antibody responses similar to bom FLOspA and FLOspB based vaccine formulated with QS-21 (p <0.2 and p <0.1, respectively). All antisera to vaccines formulated with nonlipidated or lipoproteins OspA, OspB, and QS-21 contained antibodies to bodi OspA and OspB. The representative immunoblot showed mat all 11 antisera to vaccine containing 25 μg each of FLOspA and FLOspB and 50 μg of QS-21 reacted with both OspA and OspB protein bands (Fig. 13, lane 13 to 23). OspA and OspB specific antibodies in naturally exposed canine sera Sixty three serum samples from naturally exposed dogs were tested positive for antibodies to B. burgdorferi by ELISA and immunoblotting. The representative patterns of immunoblot widi these positive sera were shown in Fig. 13 (lanes 2 to 12). Among ese positive sera (Table 1), 24 (38%) sera reacted with both OspA and OspB (Fig. 13, lane 2-7); 3 (5%) sera reacted with OspA, not OspB (lane 8) and 4 (6%) sera recognized OspB, not OspA (Lane 9-10). Most of OspA and OspB bands were faint. Thirty-two positive sera (51 %) reacted with neither OspA nor OspB (Fig. 13, lane 11-12). OspA and OspB specific IgGl and IgG2 antibody titers of these positive sera were low (Fig. 14). Most of them had antibody titers of ≤ 100. Only one serum sample had high titer of IgGl and IgG2. In contrast, antisera elicited by the experimental vaccine containing 25 μg each of FLOspA, FLOspB and QS-21 had significantly (p< 0.001) higher titer than naturally exposed dogs sera. (Fig. 14). Antiborrelial activity of canine sera The antiborrelial activity of the canine antisera to me experimental Lyme disease vaccines was tested against die homologous strain B31 and heterologous strain CA-2-87, as well as the other Borrelial species G25 and 24008 Fr. Antisera to QS-21 formulated vaccine containing TOspA and TOspB were antiborrelial to the homologous strain B31 and heterologous strain CA-2-87. Their antiborrelial titers, however, were 8-fold (p < 0.005) and 3-fold (p < 0.001) lower against B31 and CA-2-87, respectively, than those of antisera to the same formulation containing FLOspA and FLOspB (Fig. 15). Antisera to vaccine containing 25 μg each of FLOspA, FLOspB, and QS-21 possessed similar antiborrelial activity to those sera induced by vaccine containing 100 μg each of FLOspA, FLOspB, and QS-21 (Fig. 15). These antisera had significantly higher antiborrelial activity against homologous and heterologous Lyme disease spirochetes than antisera to the vaccines formulated without QS-21 or formulated with only either FLOspA or FLOspB, as indicated by higher absorbance (Fig. 16). Only these antisera to FLOspA, FLOspB, and QS-21 were also antiborrelial to B. garinii sp. nov. strain G25 and borrelial strain 24008 Fr, both European isolates (Fig. 16). On the basis of antiborrelial activity against strain B31, antiborrelial activity of the antisera correlated with isotype IgG2 antibody titer (R=0.61) better than witii IgGl antibody titer (R=0.13) (Fig. 17). Among 63 naturally exposed, seropositive dog sera, 13 (21 %) had antiborrelial activity against strain B31 (titer 10 to 160) (Table 1). Six of these 13 samples had antibodies to both OspA and OspB, and 7 samples had antibodies neitiier to OspA nor to OspB, as determined by immunoblotting. Example 4 Impact of the Saponin Adjuvant QS-21 and Aluminum Hydroxide on the Immunogenicity of Recombinant OspA and OspB of Borrelia burgdorferi Materials and Methods Bacterial strains and growth conditions Borrelia burgdorferi strains B31, CA-2-87, Fr, and G25 are described in Example 1 and in the literature (see Schwan et al, J. Clin. Microbiol. 26:557-558 (1988)). Spirochetes were grown in modified Barbour-Stoenner- Kelly (mBSK) medium at 32 °C as described in die literature (see Callister et al, J. Clin. Microbiol 25:363-365 (1990)). Molecular cloning, expression, and purification of OspA and OspB The genes encoding strain B31 OspA and OspB were cloned using published DNA sequence (Bergstrom, S. et al , Mol Microbiol. 5:479-486 (1989)) and standard molecular biological techniques (Maniatis, T. et al , Molecular Cloning, Cold Spring Harbor Laboratory, New York (1982)). Recombinant OspA starting at amino acid 17 and OspB starting at amino acid 20 were expressed in E. coli strain MZ-1 using expression vector pLCBCl (Beltz, G. A. et al. , U.S. Patent No. 4,753,873 (1988)), a vector very similar to that described by Lautenberger (Lautenberger, J. A. et al. , Gene Anal Techn. 7:63-66 (1984)) and used to express recombinant proteins to high level (Lautenberger, J. A. et al. , Gene 25:75-84 (1983)). The vector makes use of die heat inducible pL promoter; the Shine-Dalgarno sequence and first N- terminal 16 amino acid codons are derived from the Lambda ell gene. Cloning sites within three codons of mature OspA and OspB were chosen for ligation into tiie expression vector. The cloned ospA and ospB genes were confirmed by DNA sequencing (Bergstrom, S. et al. , Mol. Microbiol 5:479- 486 (1989)). Expression of OspA and OspB was confirmed widi specific monoclonal antibodies to OspA and OspB of strain B31 and polyclonal antiserum to the lysates of strain B31 as described in Examples 1 and 2 (also see Ma and Coughlin, J. Microbiol. Methods 77:145-153 (1993)). OspA and OspB were purified by reversible citraconylation and/or anion exchange chromatography as described previously (Marciani D. J. et al. , Vaccine 9:89- 96 (1991); Marciani, D. J. et al , "Solubilization of inclusion body proteins by reversible N-acylation, " in Protein Purification, R. Burgess, ed., Alan R. Liss, Inc., New York (1987), pp. 443-458). Whole Cell Lysates Borrelia burgdorferi culture at late logarithmic growth phase was centrifuged for 10 min at 10,000 xg, and die pellet washed 3 times with sterile 0.1 M phosphate-buffered saline, pH 7.2 (PBS). The pellet was resuspended in PBS containing 0.5% sodium dodecyl sulfate (SDS) and designated as whole cell lysates for immunoblotting analysis. Protein concentration of the lysates was determined by BCA assay (Pierce, Rockford, IL). Adjuvants, Vaccine Formulations, and Antisera QS-21 was purified from the cortex of the tree Quillaja saponaria monlina by ultrafiltration, adsorption chromatography, and reverse phase HPLC (Kensil, C. R. et al , J. Immunol. 746:431-437 (1990)). For mouse antiserum production, each dose (0.2 ml) of the experimental vaccine contained 25 μg of recombinant protein. This antigen dose used was found to induce significantly higher titers of antibody than either 1 μg or 5 μg of antigen (data not shown). Formulated vaccines contained eitiier 20 μg of QS- 21 or 200 μg of alum (Alhydrogel, Accurate Chemical and Scientific Corp., Westbury, NY) in PBS, pH 6.5. The adjuvant doses of both QS-21 and alum were previously optimized for the water soluble protein antigen, ovalbumin in mice (Kensil, C. R. et al , "The use of Stimulon™ to boost vacine response," Vaccine Res. 2:273-281 (1993).) For immunoprotection studies, the vaccine contained 25 μg each of OspA and OspB formulated with 20 μg of QS-21. Antisera to OspA and OspB were prepared by subcutaneous immunization of 6 to 7 week old female C3H/HeJ mice (The Jackson Laboratory, Bar Harbor, ME) 3 times at 2 week interval as described in Example 1 (also see Fikrig, E. et al , Science 250:553-556 (1990). SDS-polyacrylamide Gel Electrophoresis and Immunoblotting SDS- polyacrylamide gel electrophoresis (SDS-PAGE) was performed using an 11 % separating gel (Laemmli, U. K., Nature (London) 227:680-685 (1970)). The gel was stained witii Coomassie Brilliant blue R-250. Immunoblotting was conducted by modification of the procedure of Towbin et al. (Towbin, H. et al , Proc. Natl. Acad. Sci. USA 76:4350-5354 (1979)). Bacterial lysates (4 to 6 μg) were subjected to SDS-PAGE, transferred onto nitrocellulose membrane, and probed with both 1:20 and 1:200 dilutions of mouse antisera. Goat anti-mouse IgG conjugated to horseradish peroxidase (Fisher Scientific, Pittsburgh, PA) and 4-chloro-l-naphthol substrate was used to detect antigen-antibody binding on immunoblots. Enzyme-linked Immunosorbent Assay Appropriate concentration of each of the reagents used in die enzyme- linked immunosorbant assay (ELISA) was determined by checkerboard titration. The assay was performed using plates coated with B. burgdorferi antigens (Cambridge Biotech Corp., Worcester, MA) as previously described (Lindenmayer, J. et al , J. Clin. Microbiol 5:92-96 (1990)). For determination of antibody isotypes, an appropriate dilution of isotype-specific goat anti-mouse IgG conjugated to horseradish peroxidase (Fisher Scientific, Pittsburgh, PA) was used. Antibody titer was defined as the highest serum dilutions resulting in an absorbance value close to 1.0 in this assay. Microaggultination Borrelia burgdorferi culture was adjusted to a concentration which contained approximately 60 organisms per microscopic field. One hundred μl of B. burgdorferi was incubated in 96-well plates witii an equal volume of heat- inactivated, serially diluted antisera or normal serum in mBSK medium at 32 °C for 2 hours. Agglutination was determined by dark-field microscopy. For each assay, 3 samples (10 μl) were taken and 9 fields of each sample were examined for individual spirochetes. The number of individual spirochetes per microscopic field was averaged. Agglutination titer was defined as the highest dilution of the antisera that produced less than 15 individual spirochetes per microscopic field (i.e. caused > 50% of the spirochetes to agglutinate). Colorimetric borreliacidal Assay The colorimetric borreliacidal assay was performed in duplicate in 96- well plates (Costar, Cambridge, MA) by the method of the present invention and as described in Example 1 of die present specification. Briefly, heat- inactivated, filter-sterilized test sera were serially diluted in 100 μl of mBSK containing 120 μg/ml of phenol red (SIGMA, St. Louis, Missouri) (mBSK- PR). Five μl of sterile guinea pig complement (276 CΗ 50 /ml) (GIBCO, Grand Island, New York) and 95 μl of spirochete suspension (containing approximately 4 x 10 6 of spirochetes at logarithmic growth phase) in fresh mBSK were added to each well. After 48 to 72 h incubation at 32 °C, the absorbance at 562/630 nm was measured using a microplate reader. The titer of borreliacidal activity was defined as die highest dilution of die antisera which inhibited absorbance change caused by 50% (2 x 10 6 bacteria) of spirochetes used in the assay. For each assay, positive and negative sera were also tested for controls. Immunoprotection Studies Groups of 10 C3H/HeJ mice were subcutaneously immunized two or three times with the experimental vaccine containing OspA and OspB formulated with QS-21. Control mice were injected with sterile 0.15 M saline. Two weeks after last immunization, mice were challenged intradermally with 10 3 , 10 4 , and 10 5 infectious spirochetes of strains B31 or CA-2-87, respectively. In a separate experiment, all non-vaccinated mice were infected with as few as 10 spirochetes of either strain. Mice were sacrificed two weeks following the challenge, and the bladder, heart and blood samples were cultured for spirochetes as described (Schwan, T. G. et al , J. Clin. Microbiol 26:557-558 (1988)). Heparinized blood and the organ extracts, each in a volume of 0.5 ml, were added to separate tubes containing 6 ml of BSK medium and incubated at 32 °C. cultures were monitored for the presence of motile spirochetes every 2-3 weeks, using an acridine orange staining procedure and fluorescence microscopy as previously described (Pavia, C. S. et al , J. Infect. Dis. 163:656-659 (1991)). Statistical Analysis Two-tailed Student's t-tests were performed to compare antibody response to various groups. Significant levels were assigned assuming equal variance about the means using Microsoft Excel 4.0 for Macintosh. Results Production of Recombinant OspA and OspB Expression of OspA and OspB in E. coli was induced by a shift of incubation temperature from 32°C to 42°C (lane 2 and 4, respectively, Fig. 18). Recombinant OspA and OspB were purified from E. coli (lane 3 and 5, respectively, Fig. 18), and confirmed by immunoblotting using monoclonal antibodies to OspA and OspB of strain B31. A few faint bands with low molecular weights in lanes 3 and 5 also reacted with the monoclonal antibodies on immunoblotting (data not shown), and are therefore not E. coli contaminants. Neither OspA and OspB were acylated (data not shown). Electrophoretic and Antigenic Analysis Different geographic isolates of B. burgdorferi were electrophoretically analyzed for protein composition. All 4 strains tested were similar to each other in the electrophoretic profile of the proteins except OspA and OspB (Fig. 19A). Strains B31 and Fr produced an approximately 31 Kda OspA and 34 Kda OspB (lane 1 and 3, Fig. 19A), respectively. Whereas, strain CA-2- 87 produced an approximately 31 KDa OspA and 34.5 KDa OspB (lane 2, Fig. 19A); strain G25 produced an approximately 32 KDa OspA and 33.5 KDa OspB (lane 4, Fig. 19A). Immunoblotting of whole cell lysates of these 4 strains with antisera to OspA and OspB of strain B31 revealed that strains B31, CA-2-87, Fr, and G25 produced cross-reactive OspA and OspB. Antisera to OspB also reacted witii an approximately 22 KDa protein band of strains B31, CA-2-87, and Fr (Fig. 19C). Effect of Adjuvants on Antibody Responses to OspA and OspB Antisera from mice immunized with OspA and OspB alone or formulated witii either adjuvant QS-21 or alum were assayed for antibody responses. Isotypes IgM, IgGl, IgG2a, IgG2b, and IgG3 were measured by ELISA. Bodi OspA and OspB formulated without an adjuvant induced low titers of antibody response with the major isotype being IgGl (Fig. 20). Titers of antibody response to OspA alone were significantly higher than those to OspB. QS-21 significantly enhanced the antibody responses of IgG2a and IgG2b to OspA (p < < 0.0001 for both) and OspB (p< 0.002 and 0.02, respectively) (Fig. 20). QS-21 also significantly augmented the IgGl antibody response to OspA (p < 0.01), but not to OspB. Alum had no significant effect on IgG2a and IgG2b responses to OspB (p>0.2), but significantly inhibited IgG2a and IgG2b antibody responses to OspA (p <0.05 and p < 0.002, respectively) (Fig. 20, part A and part C). The alum formulated vaccines enhanced IgGl responses to OspB (p<0.01) but not to OspA. Vaccines formulated with both QS-21 and alum augmented the antibody responses over those induced by alum alone, but less tiian QS-21 alone. IgM and IgG3 antibody responses to OspA or OspB formulated with either QS-21 or alum were minimal when compared with other IgG isotype antibody responses. As a result, it was not possible to detect an adjuvant effect on either IgM or IgG3 induction. Microagglutination Antisera to OspA formulated with QS-21 agglutinated not only the homologous New York strain B31, but also the heterologous California isolate CA-2-87 and German isolate Fr with agglutination titers of 1600 to 6400 (Table 1). This antisera also agglutinated die Swedish isolate G25 with a titer of 400. Antisera from mice vaccinated with OspA formulated with alum or without an adjuvant had agglutination titers of < 50 to 1600. Antisera to OspB formulated with alum or QS-21 or without an adjuvant had much lower agglutination titers (< 50 to 400) with the strains tested. Borreliacidal Activity of Antisera The representative results of the borreliacidal activity of antisera to OspA and OspB are shown in Table 2. Antisera to OspA formulated without an adjuvant had low borreliacidal activity against strains B31, Fr, and G25 (titers of 40 to 80), and had undetectable activity against strain CA-2-87 (titer of < 10). QS-21 enhanced the borreliacidal anti-OspA response 8- to 64-fold as tested against these 4 strains (titers of 320 to 1280). Alum increased this response by less than 2-fold against the strains tested. OspB formulated witii either QS-21 or alum induced a 16-fold increase in borreliacidal activity against strain B31 when compared with OspB alone. A 16 fold increase in borreliacidal activity against strain Fr was also seen with antisera to OspB when formulated with QS-21, but not when formulated with alum. Neither of the antisera had borreliacidal activity against strain CA-2-87 or G25. lmmunoprotection Vaccinated mice were protected from infection following challenge with as many as 10 5 infectious spirochetes (at least 4 log greater than the infectious dose) of either the strains B31 or CA-2-87. All vaccinated mice were free of spirochetes in bladder, heart, and blood. In contrast, all control mice were infected after challenge as shown by culturing spirochete from bladder tissue samples. Some control mice also had culture positive blood and heart tissue. The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made tiiereto without departing from the spirit or scope of the invention as set forth below. All patents and publications cited herein are fully incorporated by reference herein in their entirety. INDICATIONS RELATING TO A DEPOSITED MICROORGANISM A. The indications made below relate to the microorganism referred to in the description on page \ , line ζ-) B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet | | Name of depositary institution American Type Culture Collection Address of depositary institution (including postal code and country) 12301 Parklawn Drive Rockville, Maryland 20852 United States of America Date of deposit Accession Number 25 February 1994 69577 C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet | | PLCBC10spA8+6 D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States) E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) The indications listed below will be submitted to the International Bureau later (specify the general nature of the indications e.g., "Accession Number of Deposit") For International Bureau use only I I This sheet was received by the International Bureau on: Authorized officer INDICATIONS RELATI NG TO A DEPOSITED MICROORGANISM A. The indications made below relate to the microorganism referred to in the description on page 4°1 . line QJ_ B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet | | Name of depositary institution American Type Cul ture Col lection Address of depositary institution (including postal code and country) 12301 Parkl awn Drive Rockvi l l e , Maryl and 20852 Uni ted State s of America Date of deposit Accession Number 25 February 1994 69578 C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet | | pLCBC10spB8+4 D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States) E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) The indications listed below will be submitted to the International Bureau later (specify the general nature of the indications e.g., "Accession Number of Deposit") For International Burςau use only I I This sheet was received by the International Bureau on: Authorized officer

Claims: What Is Claimed Is: 1. A method for detecting the ability of an antiserum to B. burgdoferi to kill or inhibit the growth of B. burgdorferi, which comprises (a) contacting said antiserum with B. burgdorferi and a suitable color pH indicator for measurement of bacterial growth in a medium which is compatible for the growth of B. burgdorferi in the absence of said antiserum; and (b) measuring the absorbance of the sample; wherein high absorbance, when compared to a control sample which does not contain said antiserum is an indication of high bactericidal activity. 2. The method of claim 1, wherein said color pH indicator is phenol red. 3. A method for detection of Lyme borreliosis, comprising (a) contacting an antiserum against B. burgdorferi with a suitable color pH indicator for measurement of bacterial growth, and B. burgdorferi in a medium which is compatible for the growth of B. burgdorferi in the absence of said antiserum; and (b) measuring the absorbance of the sample; wherein high absorbance, when compared to a control sample which does not contain B. burgdorferi is an indication that B. burgdorferi is present in said suspected sample. 4. The method of claim 3, wherein said color pH indicator is phenol red. 5. A vaccine comprising OspA, OspB or fragments thereof; and a saponin adjuvant. 6. The vaccine of claim 5, further comprising a pharmaceutically acceptable carrier. 7. The vaccine of claim 5, further comprising a non-saponin adjuvant. 8. The vaccine of claim 5, wherein said saponin is QS-21. 9. The vaccine of claim 5, wherein said OspA and OspB are full length and lipidated. 10. The vaccine of claim 9, wherein said saponin is QS-21. 11. The vaccine of claim 9, wherein both the lipidated full-length OspA and the lipidated full-length OspB are present. 12. The vaccine of claim 11, wherein said saponin is QS-21. 13. A method of inducing immunity to bacteria causing Lyme disease in an animal, comprising administering to the animal a vaccine comprising OspA, OspB or fragments thereof; and a saponin adjuvant. 14. The method of claim 13, wherein said saponin is QS-21. 15. The method of claim 13, wherein said OspA and OspB are full length and lipidated. 16. The method of claim 15, wherein said saponin is QS-21. 17. The method of claim 15, wherein both lipidated full-length OspA and the lipidated full-length OspB are present in said vaccine. 18. The method of claim 17, wherein said saponin is QS-21. 19. The method of claim 13, wherein said bacteria are B. burgdorferi. 20. The method of claim 13, wherein said bacteria are B. garinii. 21. The method of claim 13, wherein said B. burgdorferi bacteria are strains CA-2-87 or B-31. 22. The method of claim 20, wherein said B. garinii is strain G-25. 23. The method of claim 13, wherein said bacteria are 24008 Fr.

Inventors: COUGHLIN RICHARD T, MA JIANNENG, MARCIANI DANTE J, COUGHLIN, RICHARD, T., MA, JIANNENG, MARCIANI, DANTE, J.
Applicants: CAMBRIDGE BIOTECH CORP, CAMBRIDGE BIOTECH CORPORATION
Application year: 1994
Main classification: G01N33
IPC codes: A61K39, C07K14, C07K16, C12Q1